WorldWideScience

Sample records for clustered living cells

  1. Real-time dynamics of RNA Polymerase II clustering in live human cells

    Science.gov (United States)

    Cisse, Ibrahim

    2014-03-01

    Transcription is the first step in the central dogma of molecular biology, when genetic information encoded on DNA is made into messenger RNA. How this fundamental process occurs within living cells (in vivo) is poorly understood,[1] despite extensive biochemical characterizations with isolated biomolecules (in vitro). For high-order organisms, like humans, transcription is reported to be spatially compartmentalized in nuclear foci consisting of clusters of RNA Polymerase II, the enzyme responsible for synthesizing all messenger RNAs. However, little is known of when these foci assemble or their relative stability. We developed an approach based on photo-activation localization microscopy (PALM) combined with a temporal correlation analysis, which we refer to as tcPALM. The tcPALM method enables the real-time characterization of biomolecular spatiotemporal organization, with single-molecule sensitivity, directly in living cells.[2] Using tcPALM, we observed that RNA Polymerase II clusters form transiently, with an average lifetime of 5.1 (+/- 0.4) seconds. Stimuli affecting transcription regulation yielded orders of magnitude changes in the dynamics of the polymerase clusters, implying that clustering is regulated and plays a role in the cells ability to effect rapid response to external signals. Our results suggest that the transient crowding of enzymes may aid in rate-limiting steps of genome regulation.

  2. The complex lives of star clusters

    CERN Document Server

    Stevenson, David

    2015-01-01

    As with the author’s recent books Extreme Explosions and Under a Crimson Sun, the complex topic of star clusters is broken down and made accessible with clear links to other areas of astronomy in a language which the non-specialist can easily read and enjoy. The full range of a star cluster's lifespan is depicted, as both globular and open clusters are tracked from birth to eventual death. Why is it some are dense conglomerates of stars while others are looser associations? Are the young, brilliant clusters seen in neighboring galaxies such as the Large Magellanic Cloud, M33 or M82 analogous to the ancient globulars seen in the Milky Way? How will these clusters change as their stars wane and die? More interestingly, how does living in a dense star cluster affect the fates of the stars and any attendant planets that accompany them?   Star clusters form many of the most dazzling objects in the astronomers’ catalogs. Many amateur astronomers are interested in exploring how these objects are created and wh...

  3. Fluorescence live cell imaging.

    Science.gov (United States)

    Ettinger, Andreas; Wittmann, Torsten

    2014-01-01

    Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein (FP) tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate FP constructs by spinning disk confocal microscopy. © 2014 Elsevier Inc. All rights reserved.

  4. Live Dynamics of 53BP1 Foci Following Simultaneous Induction of Clustered and Dispersed DNA Damage in U2OS Cells

    Directory of Open Access Journals (Sweden)

    Alice Sollazzo

    2018-02-01

    Full Text Available Cells react differently to clustered and dispersed DNA double strand breaks (DSB. Little is known about the initial reaction to simultaneous induction of DSBs with different complexities. Here, we used live cell microscopy to analyse the behaviour of 53BP1-GFP (green fluorescence protein foci formation at DSBs induced in U2OS cells by alpha particles, X-rays or mixed beams over a 75 min period post irradiation. X-ray-induced foci rapidly increased and declined over the observation interval. After an initial increase, mixed beam-induced foci remained at a constant level over the observation interval, similarly as alpha-induced foci. The average areas of radiation-induced foci were similar for mixed beams and X-rays, being significantly smaller than those induced by alpha particles. Pixel intensities were highest for mixed beam-induced foci and showed the lowest level of variability over time as compared to foci induced by alphas and X-rays alone. Finally, mixed beam-exposed foci showed the lowest level of mobility as compared to alpha and X-ray exposure. The results suggest paralysation of chromatin around foci containing clustered DNA damage.

  5. 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.

  6. 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.

  7. Electronic Interfacing with Living Cells

    Science.gov (United States)

    Fleming, James T.

    The direct interfacing of living cells with inorganic electronic materials, components or systems has led to the development of two broad categories of devices that can (1) transduce biochemical signals generated by biological components into electrical signals and (2) transduce electronically generated signals into biochemical signals. The first category of devices permits the monitoring of living cells, the second, enables control of cellular processes. This review will survey this exciting area with emphasis on the fundamental issues and obstacles faced by researchers. Devices and applications that use both prokaryotic (microbial) and eukaryotic (mammalian) cells will be covered. Individual devices described include microbial biofuel cells that produce electricity, bioelectrical reactors that enable electronic control of cellular metabolism, living cell biosensors for the detection of chemicals and devices that permit monitoring and control of mammalian physiology.

  8. Diffusion inside living human cells

    DEFF Research Database (Denmark)

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

    2012-01-01

    Naturally occurring lipid granules diffuse in the cytoplasm and can be used as tracers to map out the viscoelastic landscape inside living cells. Using optical trapping and single particle tracking we found that lipid granules exhibit anomalous diffusion inside human umbilical vein endothelial...

  9. Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

    Science.gov (United States)

    Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

    2017-01-01

    Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited.

  10. Single Molecule Spectroscopy: Single Live Cell

    Indian Academy of Sciences (India)

    kbhattacharjee

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

  11. Livings Labs’ for New Health Concepts and Medical Technology in Cluster Development

    NARCIS (Netherlands)

    Guldemond, N.A.; Van Geenhuizen, M.S.

    2012-01-01

    The development of medical clusters is high on the agenda of many policymakers in the European Union. Constructing medical clusters may draw on the presence of living labs of different kinds in the regions involved. Living labs are practical environments for innovation aiming at a better mix and

  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. Nutrient shielding in clusters of cells

    Science.gov (United States)

    Lavrentovich, Maxim O.; Koschwanez, John H.; Nelson, David R.

    2013-06-01

    Cellular nutrient consumption is influenced by both the nutrient uptake kinetics of an individual cell and the cells' spatial arrangement. Large cell clusters or colonies have inhibited growth at the cluster's center due to the shielding of nutrients by the cells closer to the surface. We develop an effective medium theory that predicts a thickness ℓ of the outer shell of cells in the cluster that receives enough nutrient to grow. The cells are treated as partially absorbing identical spherical nutrient sinks, and we identify a dimensionless parameter ν that characterizes the absorption strength of each cell. The parameter ν can vary over many orders of magnitude among different cell types, ranging from bacteria and yeast to human tissue. The thickness ℓ decreases with increasing ν, increasing cell volume fraction ϕ, and decreasing ambient nutrient concentration ψ∞. The theoretical results are compared with numerical simulations and experiments. In the latter studies, colonies of budding yeast, Saccharomyces cerevisiae, are grown on glucose media and imaged under a confocal microscope. We measure the growth inside the colonies via a fluorescent protein reporter and compare the experimental and theoretical results for the thickness ℓ.

  14. 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.

  15. Nutrient Shielding in Clusters of Cells

    Science.gov (United States)

    Lavrentovich, Maxim O.; Koschwanez, John H.; Nelson, David R.

    2014-01-01

    Cellular nutrient consumption is influenced by both the nutrient uptake kinetics of an individual cell and the cells’ spatial arrangement. Large cell clusters or colonies have inhibited growth at the cluster's center due to the shielding of nutrients by the cells closer to the surface. We develop an effective medium theory that predicts a thickness ℓ of the outer shell of cells in the cluster that receives enough nutrient to grow. The cells are treated as partially absorbing identical spherical nutrient sinks, and we identify a dimensionless parameter ν that characterizes the absorption strength of each cell. The parameter ν can vary over many orders of magnitude between different cell types, ranging from bacteria and yeast to human tissue. The thickness ℓ decreases with increasing ν, increasing cell volume fraction ϕ, and decreasing ambient nutrient concentration ψ∞. The theoretical results are compared with numerical simulations and experiments. In the latter studies, colonies of budding yeast, Saccharomyces cerevisiae, are grown on glucose media and imaged under a confocal microscope. We measure the growth inside the colonies via a fluorescent protein reporter and compare the experimental and theoretical results for the thickness ℓ. PMID:23848711

  16. Nucleic Acid Aptamers for Living Cell Analysis

    Science.gov (United States)

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

    2014-06-01

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

  17. Methods for Screening Live Cells.

    Science.gov (United States)

    Gordeev, A A; Chetverin, A B

    2018-01-01

    Cell screening or, in other words, identification of cells with certain properties is now increasingly used in scientific and medical research, e.g., in diagnostics, drug testing, and production of cell clones with desired characteristics. In this review, we discuss existing methods of cell screening and their classification according to the cell presentation format. We describe the principles of the one-dimensional and two-dimensional formats and compare the main advantages and drawbacks of these formats. The first part describes the methods based on the 2D-format of cell presentation, when cells are immobilized in the same plane by various techniques. The second part describes the methods of the 1D-screening, when cells are aligned in a line in a stream of fluid and scanned one-by-one while passing through a detector. The final part of the review describes the method of high-performance cell analysis based on the merged gel technique. This technique combines the advantages of both 1D and 2D formats and, according to the authors, might become an effective alternative to many modern methods of cell screening.

  18. Study of ^{14}C Cluster Decay Half-Lives of Heavy Deformed Nuclei

    Science.gov (United States)

    Shamami, S. Rahimi; Pahlavani, M. R.

    2018-01-01

    A theoretical model based on deformed Woods-Saxon, Coulomb and centrifugal terms are constructed to evaluate the half-lives for the cluster radioactivity of various super heavy nuclei. Deformation have been applied on all parts of their potential containing nuclear barrier for cluster decay. Also, both parent and daughter nuclei are considered to be deformed. The calculated results of ^{14}C cluster radioactivity half-lives are compared with available experimental data. A satisfactory agreement between theoretical and measured data is achieved. Also, obtained half-lives for each decay family is agreed with Geiger-Nuttall law.

  19. Drug delivery with living cells

    NARCIS (Netherlands)

    Fliervoet, Lies A L; Mastrobattista, Enrico

    2016-01-01

    The field of drug delivery has grown tremendously in the past few decades by developing a wide range of advanced drug delivery systems. An interesting category is cell-based drug delivery, which includes encapsulation of drugs inside cells or attached to the surface and subsequent transportation

  20. Ethnic Clusters in Public Housing and Independent Living of Elderly Immigrants from the Former Soviet Union.

    Science.gov (United States)

    Vinokurov, Andrey; Trickett, Edison J

    2015-12-01

    The study examines the effects of ethnic clusters and independent living arrangements on adaptation of elderly immigrants from the Former Soviet Union. The multigenerational living arrangements were compared with independent living in a dispersed ethnic community and in an ethnic cluster of public housing. The residents of the ethnic clusters of public housing reported poorer health, were more reliant on government resources, and experienced greater acculturative hassles. However, public housing residents reported significantly larger Russian-speaking and American social networks, greater American acculturation, higher social support from neighbors, as well as lower cultural alienation. In contrast, the multigenerational living arrangements were related to greater social support from extended family and higher extended family satisfaction. While, the independent living in the dispersed ethnic community was associated with smaller American social networks and higher levels of cultural alienation. The results highlight how the ecologies of different living arrangements are reflected in the nature of acculturative, social, and psychological experiences of elderly immigrants.

  1. Live Cell Imaging in Fission Yeast.

    Science.gov (United States)

    Mulvihill, Daniel P

    2017-10-03

    Live cell imaging complements the array of biochemical and molecular genetic approaches to provide a comprehensive insight into functional dependencies and molecular interactions in fission yeast. Fluorescent proteins and vital dyes reveal dynamic changes in the spatial distribution of organelles and the proteome and how each alters in response to changes in environmental and genetic composition. This introduction discusses key issues and basic image analysis for live cell imaging of fission yeast. © 2017 Cold Spring Harbor Laboratory Press.

  2. Ionic strength sensing in living cells

    NARCIS (Netherlands)

    Liu, Boqun; Poolman, Bert; Boersma, Arnold J

    Knowledge of the ionic strength in cells is required to understand the in vivo biochemistry of the charged biomacromolecules. Here, we present the first sensors to determine the ionic strength in living cells, by designing protein probes based on Förster resonance energy transfer (FRET). These

  3. 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.

  4. Cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells.

    Science.gov (United States)

    Castiel, Asher; Visochek, Leonid; Mittelman, Leonid; Zilberstein, Yael; Dantzer, Francoise; Izraeli, Shai; Cohen-Armon, Malka

    2013-08-21

    Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent γ-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered γ-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

  5. Dynamics of proteasome distribution in living cells

    NARCIS (Netherlands)

    Reits, E. A.; Benham, A. M.; Plougastel, B.; Neefjes, J.; Trowsdale, J.

    1997-01-01

    Proteasomes are proteolytic complexes involved in non-lysosomal degradation which are localized in both the cytoplasm and the nucleus. The dynamics of proteasomes in living cells is unclear, as is their targeting to proteins destined for degradation. To investigate the intracellular distribution and

  6. 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.

  7. Radiobiological analyse based on cell cluster models

    International Nuclear Information System (INIS)

    Lin Hui; Jing Jia; Meng Damin; Xu Yuanying; Xu Liangfeng

    2010-01-01

    The influence of cell cluster dimension on EUD and TCP for targeted radionuclide therapy was studied using the radiobiological method. The radiobiological features of tumor with activity-lack in core were evaluated and analyzed by associating EUD, TCP and SF.The results show that EUD will increase with the increase of tumor dimension under the activity homogeneous distribution. If the extra-cellular activity was taken into consideration, the EUD will increase 47%. Under the activity-lack in tumor center and the requirement of TCP=0.90, the α cross-fire influence of 211 At could make up the maximum(48 μm)3 activity-lack for Nucleus source, but(72 μm)3 for Cytoplasm, Cell Surface, Cell and Voxel sources. In clinic,the physician could prefer the suggested dose of Cell Surface source in case of the future of local tumor control for under-dose. Generally TCP could well exhibit the effect difference between under-dose and due-dose, but not between due-dose and over-dose, which makes TCP more suitable for the therapy plan choice. EUD could well exhibit the difference between different models and activity distributions,which makes it more suitable for the research work. When the user uses EUD to study the influence of activity inhomogeneous distribution, one should keep the consistency of the configuration and volume of the former and the latter models. (authors)

  8. Clusters of Circulating Tumor Cells: a Biophysical and Technological Perspective.

    Science.gov (United States)

    Au, Sam H; Edd, Jon; Haber, Daniel A; Maheswaran, Shyamala; Stott, Shannon L; Toner, Mehmet

    2017-09-01

    The vast majority of cancer associated deaths result from metastasis, yet the behaviors of its most potent cellular driver, circulating tumor cell clusters, are only beginning to be revealed. This review highlights recent advances to our understanding of tumor cell clusters with emphasis on enabling technologies. The importance of intercellular adhesions among cells in clusters have begun to be unraveled with the aid of promising microfluidic strategies for isolating clusters from patient blood. Due to their metastatic potency, the utility of circulating tumor cell clusters for cancer diagnosis, drug screening, precision oncology and as targets of antimetastatic therapeutics are being explored. The continued development of tools for exploring circulating tumor cell clusters will enhance our fundamental understanding of the metastatic process and may be instrumental in devising new strategies to suppress and eliminate metastasis.

  9. 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.)

  10. 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.

  11. Tumor-derived circulating endothelial cell clusters in colorectal cancer.

    KAUST Repository

    Cima, Igor

    2016-06-29

    Clusters of tumor cells are often observed in the blood of cancer patients. These structures have been described as malignant entities for more than 50 years, although their comprehensive characterization is lacking. Contrary to current consensus, we demonstrate that a discrete population of circulating cell clusters isolated from the blood of colorectal cancer patients are not cancerous but consist of tumor-derived endothelial cells. These clusters express both epithelial and mesenchymal markers, consistent with previous reports on circulating tumor cell (CTC) phenotyping. However, unlike CTCs, they do not mirror the genetic variations of matched tumors. Transcriptomic analysis of single clusters revealed that these structures exhibit an endothelial phenotype and can be traced back to the tumor endothelium. Further results show that tumor-derived endothelial clusters do not form by coagulation or by outgrowth of single circulating endothelial cells, supporting a direct release of clusters from the tumor vasculature. The isolation and enumeration of these benign clusters distinguished healthy volunteers from treatment-naïve as well as pathological early-stage (≤IIA) colorectal cancer patients with high accuracy, suggesting that tumor-derived circulating endothelial cell clusters could be used as a means of noninvasive screening for colorectal cancer. In contrast to CTCs, tumor-derived endothelial cell clusters may also provide important information about the underlying tumor vasculature at the time of diagnosis, during treatment, and throughout the course of the disease.

  12. 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.

  13. Optical magnetic imaging of living cells

    Science.gov (United States)

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

    2013-01-01

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

  14. 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.

  15. 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

  16. Raster image correlation spectroscopy in live cells.

    Science.gov (United States)

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

    2010-11-01

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

  17. Reconfigurable Analog Signal Processing by Living Cells.

    Science.gov (United States)

    Lewis, Daniel D; Chavez, Michael; Chiu, Kwan Lun; Tan, Cheemeng

    2018-01-19

    Living cells are known for their capacity for versatile signal processing, particularly the ability to respond differently to the same stimuli using biochemical networks that integrate environmental signals and reconfigure their dynamic responses. However, the complexity of natural biological networks confounds the discovery of fundamental mechanisms behind versatile signaling. Here, we study one specific aspect of reconfigurable signal processing in which a minimal biological network integrates two signals, using one to reconfigure the network's transfer function with respect to the other, producing an emergent switch between induction and repression. In contrast to known mechanisms, the new mechanism reconfigures transfer functions through genetic networks without extensive protein-protein interactions. These results provide a novel explanation for the versatility of genetic programs, and suggest a new mechanism of signal integration that may govern flexibility and plasticity of gene expression.

  18. Dynamical properties of water in living cells

    Science.gov (United States)

    Piazza, Irina; Cupane, Antonio; Barbier, Emmanuel L.; Rome, Claire; Collomb, Nora; Ollivier, Jacques; Gonzalez, Miguel A.; Natali, Francesca

    2018-02-01

    With the aim of studying the effect of water dynamics on the properties of biological systems, in this paper, we present a quasi-elastic neutron scattering study on three different types of living cells, differing both in their morphological and tumor properties. The measured scattering signal, which essentially originates from hydrogen atoms present in the investigated systems, has been analyzed using a global fitting strategy using an optimized theoretical model that considers various classes of hydrogen atoms and allows disentangling diffusive and rotational motions. The approach has been carefully validated by checking the reliability of the calculation of parameters and their 99% confidence intervals. We demonstrate that quasi-elastic neutron scattering is a suitable experimental technique to characterize the dynamics of intracellular water in the angstrom/picosecond space/time scale and to investigate the effect of water dynamics on cellular biodiversity.

  19. 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

  20. Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution

    Science.gov (United States)

    Sabet, Ola; Wehner, Frank; Konitsiotis, Antonios; Fuhr, Günther R.; Bastiaens, Philippe I. H.

    2016-01-01

    The dynamics of molecules in living cells hamper precise imaging of molecular patterns by functional and super resolution microscopy. Circumventing lethal chemical fixation, an on-stage cryo-arrest was developed for consecutive imaging of molecular patterns within the same living, but arrested cells. The reversibility of consecutive cryo-arrests was demonstrated by the high survival rate of different cell lines and intact growth factor signaling that was not perturbed by stress response. Reversible cryo-arrest was applied to study the evolution of ligand-induced receptor tyrosine kinase activation at different scales. The nanoscale clustering of epidermal growth factor receptor (EGFR) in the plasma membrane was assessed by single molecule localization microscopy and endosomal microscale activity patterns of ephrin receptor type-A (EphA2) by fluorescence lifetime imaging microscopy. We thereby demonstrate that reversible cryo-arrest allows the precise determination of molecular patterns while conserving the dynamic capabilities of living cells. PMID:27400419

  1. 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.

  2. Immunolabeling artifacts and the need for live-cell imaging

    NARCIS (Netherlands)

    Schnell, Ulrike; Dijk, Freark; Sjollema, Klaas A.; Giepmans, Ben N. G.

    Fluorescent fusion proteins have revolutionized examination of proteins in living cells. Still, studies using these proteins are met with criticism because proteins are modified and ectopically expressed, in contrast to immunofluorescence studies. However, introducing immunoreagents inside cells can

  3. Clustering of red blood cells using digital holographic microscopy

    Science.gov (United States)

    Jaferzadeh, K.; Ahmadzadeh, E.; Moon, I.; Gholami, S.

    2017-05-01

    Digital holographic microscopy can provide quantitative phase images (QPIs) of 3D profile of red blood cell (RBC) with nanometer accuracy. In this paper we propose applying k-means clustering method to cluster RBCs into two groups of young and old RBCs by using a four-dimensional feature vector. The features are RBC thickness average, surface area-volume ratio, sphericity coefficient and RBC perimeter that can be obtained from QPIs. The proposed features are related to the morphology of RBC. The experimental result shows that by utilizing the proposed method two groups of sphero-echinocytes (old RBCs) and non-spheroechinocytes RBCs can be perfectly clustered.

  4. Live Imaging of the Drosophila Testis Stem Cell Niche.

    Science.gov (United States)

    Greenspan, Leah J; Matunis, Erika L

    2017-01-01

    Live imaging of adult tissue stem cell niches provides key insights into the dynamic behavior of stem cells, their differentiating progeny, and their neighboring support cells, but few niches are amenable to this approach. Here we discuss a technique for long-term live imaging of the Drosophila testis stem cell niche. Culturing whole testes ex vivo for up to 12.5 h allows for tracking of cell-type specific behaviors under normal and various chemically or genetically modified conditions. Fixing and staining tissues after live imaging allows for the molecular confirmation of cell identity and behavior. Utilization of live imaging in intact niches will facilitate further understanding of the cellular and molecular mechanisms that regulate stem cell function in vivo.

  5. Triggered release kinetics of living cells from composite microcapsules

    NARCIS (Netherlands)

    Hamad, S.A.; Stoyanov, S.D.; Paunov, V.N.

    2013-01-01

    We have developed a theoretical model for the kinetics of release of living cells from composite shellac-cell microcapsules. The model describes the kinetics of cell release from the microcapsules triggered by: (i) pH change, which dissolves the shellac and (ii) the growth of the encapsulated cells,

  6. p53 Dependent Centrosome Clustering Prevents Multipolar Mitosis in Tetraploid Cells

    Science.gov (United States)

    Yi, Qiyi; Zhao, Xiaoyu; Huang, Yun; Ma, Tieliang; Zhang, Yingyin; Hou, Heli; Cooke, Howard J.; Yang, Da-Qing; Wu, Mian; Shi, Qinghua

    2011-01-01

    Background p53 abnormality and aneuploidy often coexist in human tumors, and tetraploidy is considered as an intermediate between normal diploidy and aneuploidy. The purpose of this study was to investigate whether and how p53 influences the transformation from tetraploidy to aneuploidy. Principal Findings Live cell imaging was performed to determine the fates and mitotic behaviors of several human and mouse tetraploid cells with different p53 status, and centrosome and spindle immunostaining was used to investigate centrosome behaviors. We found that p53 dominant-negative mutation, point mutation, or knockout led to a 2∼ 33-fold increase of multipolar mitosis in N/TERT1, 3T3 and mouse embryonic fibroblasts (MEFs), while mitotic entry and cell death were not significantly affected. In p53-/- tetraploid MEFs, the ability of centrosome clustering was compromised, while centrosome inactivation was not affected. Suppression of RhoA/ROCK activity by specific inhibitors in p53-/- tetraploid MEFs enhanced centrosome clustering, decreased multipolar mitosis from 38% to 20% and 16% for RhoA and ROCK, respectively, while expression of constitutively active RhoA in p53+/+ tetraploid 3T3 cells increased the frequency of multipolar mitosis from 15% to 35%. Conclusions p53 could not prevent tetraploid cells entering mitosis or induce tetraploid cell death. However, p53 abnormality impaired centrosome clustering and lead to multipolar mitosis in tetraploid cells by modulating the RhoA/ROCK signaling pathway. PMID:22076149

  7. Phase cell cluster expansion for phi43

    International Nuclear Information System (INIS)

    Williamson, C.

    1987-01-01

    We complete the work begun by Battle and Federbush in their approach to the phi 4 3 Euclidean field theory. In their papers, they applied a cluster expansion to a phi 4 3 interaction using a diagonal covariance C/sub i//sub j/ = δ/sub i//sub j/. In our model we take the usual covariance C = (-Δ+M 2 ) -1 . Since this is no longer diagonal we must interpolate this covariance to decouple terms. Complications in our model come from an extra term that arises during this interpolation and also from the couplings in integration-by-parts formulas between variables at different scales. The extra term that arises in interpolating the covariance leads to a chain that must sometimes be attached to different places in the tree structure of the expansion. We find more factorials from the additional attachments created doing this and use some of these to cancel a part of the number divergence that arises from the nondiagonal covariance terms in our integration-by-parts formulas. copyright 1987 Academic Press, Inc

  8. Spatial distribution and temporal evolution of DRONPA-fused SNAP25 clusters in adrenal chromaffin cells

    DEFF Research Database (Denmark)

    Antoku, Yasuko; Dedecker, Peter; da Silva Pinheiro, Paulo César

    2015-01-01

    fluorescence bursts of DRONPA-fused SNAP-25 molecules in live chromaffin cells by Total Internal Reflection Fluorescence (TIRF) imaging. We find that this method allows tracking protein cluster dynamics over relatively long times (∼20 min.), partly due to the diffusion into the TIRF field of fresh molecules......Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (Soluble NSF Attachment Protein Receptor) proteins involved in exocytosis, in fixed cells have resulted in images with high spatial resolution, at the expense of dynamical information. Here, we have imaged localized...

  9. 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.

  10. 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)

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

    Science.gov (United States)

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

    2013-05-01

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

  12. 3D imaging of Sox2 enhancer clusters in embryonic stem cells.

    Science.gov (United States)

    Liu, Zhe; Legant, Wesley R; Chen, Bi-Chang; Li, Li; Grimm, Jonathan B; Lavis, Luke D; Betzig, Eric; Tjian, Robert

    2014-12-24

    Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription.

  13. 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

  14. 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

  15. CELL FORMATION IN GROUP TECHNOLOGY: A SIMILARITY ORDER CLUSTERING APPROACH

    Directory of Open Access Journals (Sweden)

    Godfrey C. Onwubolu

    2012-01-01

    Full Text Available Grouping parts into families which can be produced by a cluster of machine cells is the cornerstone of cellular manufacturing, which in turn is the building block for flexible manufacturing systems. Cellular manufacturing is a group technology (GT concept that has recently attracted the attention of manufacturing firms operating under jobshop environment to consider redesigning their manufacturing systems so as to take advantage of increased throughput, reduction in work-in-progress, set-up time, and lead times; leading to product quality and customer satisfaction. The paper presents a generalised approach for machine cell formation from a jobshop using similarity order clustering technique for preliminary cell grouping and considering machine utilisation for the design of nonintergrouping material handling using the single-pass heuristic. The work addresses the shortcomings of cellular manufacturing systems design and implementations which ignore machine utilisations, group sizes and intergroup moves.

  16. 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.

  17. 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.

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

    Science.gov (United States)

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

    2017-05-24

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

  19. 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 ...

  20. 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.

  1. Effects of Nurse-Led Multifactorial Care to Prevent Disability in Community-Living Older People : Cluster Randomized Trial

    NARCIS (Netherlands)

    Suijker, Jacqueline J.; van Rijn, Marjon; Buurman, Bianca M.; ter Riet, Gerben; van Charante, Eric P. Moll; de Rooij, Sophia E.

    2016-01-01

    Background To evaluate the effects of nurse-led multifactorial care to prevent disability in community-living older people. Methods In a cluster randomized trail, 11 practices (n = 1,209 participants) were randomized to the intervention group, and 13 practices (n = 1,074 participants) were

  2. Effects of Nurse-Led Multifactorial Care to Prevent Disability in Community-Living Older People: Cluster Randomized Trial

    NARCIS (Netherlands)

    Suijker, Jacqueline J.; van Rijn, Marjon; Buurman, Bianca M.; ter Riet, Gerben; Moll van Charante, Eric P.; de Rooij, Sophia E.

    2016-01-01

    To evaluate the effects of nurse-led multifactorial care to prevent disability in community-living older people. In a cluster randomized trail, 11 practices (n = 1,209 participants) were randomized to the intervention group, and 13 practices (n = 1,074 participants) were randomized to the control

  3. Mapping of spatiotemporal heterogeneous particle dynamics in living cells

    NARCIS (Netherlands)

    Duits, Michael H.G.; Li, Y.; Vanapalli Veera, V.S.A.R.; Vanapalli, Srinivas; Mugele, Friedrich Gunther

    2009-01-01

    Colloidal particles embedded in the cytoplasm of living mammalian cells have been found to display remarkable heterogeneity in their amplitude of motion. However, consensus on the significance and origin of this phenomenon is still lacking. We conducted experiments on Hmec-1 cells loaded with about

  4. Live Attenuated Versus Inactivated Influenza Vaccine in Hutterite Children: A Cluster Randomized Blinded Trial.

    Science.gov (United States)

    Loeb, Mark; Russell, Margaret L; Manning, Vanessa; Fonseca, Kevin; Earn, David J D; Horsman, Gregory; Chokani, Khami; Vooght, Mark; Babiuk, Lorne; Schwartz, Lisa; Neupane, Binod; Singh, Pardeep; Walter, Stephen D; Pullenayegum, Eleanor

    2016-11-01

    Whether vaccinating children with intranasal live attenuated influenza vaccine (LAIV) is more effective than inactivated influenza vaccine (IIV) in providing both direct protection in vaccinated persons and herd protection in unvaccinated persons is uncertain. Hutterite colonies, where members live in close-knit, small rural communities in which influenza virus infection regularly occurs, offer an opportunity to address this question. To determine whether vaccinating children and adolescents with LAIV provides better community protection than IIV. A cluster randomized blinded trial conducted between October 2012 and May 2015 over 3 influenza seasons. (ClinicalTrials.gov: NCT01653015). 52 Hutterite colonies in Alberta and Saskatchewan, Canada. 1186 Canadian children and adolescents aged 36 months to 15 years who received the study vaccine and 3425 community members who did not. Children were randomly assigned according to community in a blinded manner to receive standard dosing of either trivalent LAIV or trivalent IIV. The primary outcome was reverse transcriptase polymerase chain reaction-confirmed influenza A or B virus in all participants (vaccinated children and persons who did not receive the study vaccine). Mean vaccine coverage among children in the LAIV group was 76.9% versus 72.3% in the IIV group. Influenza virus infection occurred at a rate of 5.3% (295 of 5560 person-years) in the LAIV group versus 5.2% (304 of 5810 person-years) in the IIV group. The hazard ratio comparing LAIV with IIV for influenza A or B virus was 1.03 (95% CI, 0.85 to 1.24). The study was conducted in Hutterite communities, which may limit generalizability. Immunizing children with LAIV does not provide better community protection against influenza than IIV. The Canadian Institutes for Health Research.

  5. Microfluidic Isolation of Circulating Tumor Cell Clusters by Size and Asymmetry.

    Science.gov (United States)

    Au, Sam H; Edd, Jon; Stoddard, Amy E; Wong, Keith H K; Fachin, Fabio; Maheswaran, Shyamala; Haber, Daniel A; Stott, Shannon L; Kapur, Ravi; Toner, Mehmet

    2017-05-26

    Circulating tumor cell clusters (CTC clusters) are potent initiators of metastasis and potentially useful clinical markers for patients with cancer. Although there are numerous devices developed to isolate individual circulating tumor cells from blood, these devices are ineffective at capturing CTC clusters, incapable of separating clusters from single cells and/or cause cluster damage or dissociation during processing. The only device currently able to specifically isolate CTC clusters from single CTCs and blood cells relies on the batch immobilization of clusters onto micropillars which necessitates long residence times and causes damage to clusters during release. Here, we present a two-stage continuous microfluidic chip that isolates and recovers viable CTC clusters from blood. This approach uses deterministic lateral displacement to sort clusters by capitalizing on two geometric properties: size and asymmetry. Cultured breast cancer CTC clusters containing between 2-100 + cells were recovered from whole blood using this integrated two-stage device with minimal cluster dissociation, 99% recovery of large clusters, cell viabilities over 87% and greater than five-log depletion of red blood cells. This continuous-flow cluster chip will enable further studies examining CTC clusters in research and clinical applications.

  6. 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.

  7. 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.

  8. 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...... control cell physiology. Indeed, in the living cell homeostatic control mechanisms might exist for the free-energy transduction pathways so as to prevent perturbation of cellular function when the Gibbs energy supply is compromised. This presentation addresses the extent to which the intracellular ATP...... level is involved in the control of cell physiology, how the elaborate control of cell function may be analyzed theoretically and quantitatively, and if this can be utilized selectively to affect certain cell types....

  9. 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

  10. Studying endosomes in cultured neurons by live-cell imaging.

    Science.gov (United States)

    Lasiecka, Zofia M; Winckler, Bettina

    2016-01-01

    Endosomes play critical roles on regulating surface receptor levels as well as signaling cascades in all cell types, including neurons. Endocytosis and endosomal trafficking is routinely studied after fixation, but live imaging is increasingly being used to capture the dynamic nature of endosomes and is allowing increasingly sophisticated glimpses into trafficking processes in live neurons. In this chapter, we describe the basics of neuronal primary cultures, methods for expressing fluorescent proteins, and live imaging of cargos and endosomal regulators. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. 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

  12. Cell Migration in Tissues: Explant Culture and Live Imaging.

    Science.gov (United States)

    Staneva, Ralitza; Barbazan, Jorge; Simon, Anthony; Vignjevic, Danijela Matic; Krndija, Denis

    2018-01-01

    Cell migration is a process that ensures correct cell localization and function in development and homeostasis. In disease such as cancer, cells acquire an upregulated migratory capacity that leads to their dissemination throughout the body. Live imaging of cell migration allows for better understanding of cell behaviors in development, adult tissue homeostasis and disease. We have optimized live imaging procedures to track cell migration in adult murine tissue explants derived from: (1) healthy gut; (2) primary intestinal carcinoma; and (3) the liver, a common metastatic site. To track epithelial cell migration in the gut, we generated an inducible fluorescent reporter mouse, enabling us to visualize and track individual cells in unperturbed gut epithelium. To image intratumoral cancer cells, we use a spontaneous intestinal cancer model based on the activation of Notch1 and deletion of p53 in the mouse intestinal epithelium, which gives rise to aggressive carcinoma. Interaction of cancer cells with a metastatic niche, the mouse liver, is addressed using a liver colonization model. In summary, we describe a method for long-term 3D imaging of tissue explants by two-photon excitation microscopy. Explant culturing and imaging can help understand dynamic behavior of cells in homeostasis and disease, and would be applicable to various tissues.

  13. Measurement of diffusion of fluorescent molecules in living cells

    International Nuclear Information System (INIS)

    Tatarkova, S A; Lloyd, C; Khaira, S K; Berk, D

    2003-01-01

    The possibilities of the method of fluorescence correlation spectroscopy for studying the molecular dynamics in living cells are demonstrated. The method provides point measurements of extremely low concentrations of fluorescent molecules and their diffusion coefficients with a high time resolution in a microscopic volume, which is especially important in pharmacological investigations. A biological model of the interaction of liposomes with a cellular membrane is considered. The diffusion coefficients of fluorescent molecules are measured directly in the living cell cytoplasm. (laser applications and other topics in quantum electronics)

  14. Quantal release of ATP from clusters of PC12 cells.

    Science.gov (United States)

    Fabbro, Alessandra; Skorinkin, Andrei; Grandolfo, Micaela; Nistri, Andrea; Giniatullin, Rashid

    2004-10-15

    Although ATP is important for intercellular communication, little is known about the mechanism of endogenous ATP release due to a dearth of suitable models. Using PC12 cells known to express the P2X2 subtype of ATP receptors and to store ATP with catecholamines inside dense-core vesicles, we found that clusters of PC12 cells cultured for 3-7 days generated small transient inward currents (STICs) after an inward current elicited by exogenous ATP. The amplitude of STICs in individual cells correlated with the peak amplitude of ATP-induced currents. STICs appeared as asynchronous responses (approximately 20 pA average amplitude) for 1-20 s and were investigated with a combination of patch clamping, Ca2+ imaging, biochemistry and electron microscopy. Comparable STICs were produced by focal KCl pulses and were dependent on extracellular Ca2+. STICs were abolished by the P2X antagonist PPADS and potentiated by Zn2+, suggesting they were mediated by P2X2 receptor activation. The highest probability of observing STICs was after the peak of intracellular Ca2+ increase caused by KCl. Biochemical measurements indicated that KCl application induced a significant release of ATP from PC12 cells. Electron microscopy studies showed narrow clefts without 'synaptic-like' densities between clustered cells. Our data suggest that STICs were caused by quantal release of endogenous ATP by depolarized PC12 cells in close juxtaposition to the recorded cell. Thus, STICs may be a new experimental model to characterize the physiology of vesicular release of ATP and to study the kinetics and pharmacology of P2X2 receptor-mediated quantal currents.

  15. Characterization of Coelenterazine Analogs for Measurements of Renilla Luciferase Activity in Live Cells and Living Animals

    Directory of Open Access Journals (Sweden)

    Hui Zhao

    2004-01-01

    Full Text Available In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which analog is most suitable for activity measurements in live cells and living animals, we characterized 10 CLZN analogs using Renilla luciferase (Rluc as the reporter enzyme. For each analog, we monitored enzyme activity, auto-oxidation, and efficiency of cellular uptake. All CLZN analogs tested showed higher auto-oxidation signals in serum than was observed in phosphate buffer or medium, mainly as a result of auto-oxidation by binding to albumin. CLZN-f, -h, and -e analogs showed 4- to 8-fold greater Rluc activity, relative to CLZN-native, in cells expressing the enzyme from a stable integrant. In studies using living mice expressing Rluc in hepatocytes, administration of CLZN-e and -native produced the highest signal. Furthermore, distinct temporal differences in signal for each analog were revealed following intravenous or intraperitoneal delivery. We conclude that the CLZN analogs that are presently available vary with respect to hRluc utilization in culture and in vivo, and that the effective use of CLZN-utilizing enzymes in living animals depends on the selection of an appropriate substrate.

  16. Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins.

    Science.gov (United States)

    Hein, Birka; Willig, Katrin I; Wurm, Christian A; Westphal, Volker; Jakobs, Stefan; Hell, Stefan W

    2010-01-06

    We show far-field fluorescence nanoscopy of different structural elements labeled with an organic dye within living mammalian cells. The diffraction barrier limiting far-field light microscopy is outperformed by using stimulated emission depletion. We used the tagging protein hAGT (SNAP-tag), which covalently binds benzylguanine-substituted organic dyes, for labeling. Tetramethylrhodamine was used to image the cytoskeleton (vimentin and microtubule-associated protein 2) as well as structures located at the cell membrane (caveolin and connexin-43) with a resolution down to 40 nm. Comparison with structures labeled with the yellow fluorescent protein Citrine validates this labeling approach. Nanoscopic movies showing the movement of connexin-43 clusters across the cell membrane evidence the capability of this technique to observe structural changes on the nanoscale over time. Pulsed or continuous-wave lasers for excitation and stimulated emission depletion yield images of similar resolution in living cells. Hence fusion proteins that bind modified organic dyes expand widely the application range of far-field fluorescence nanoscopy of living cells. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. 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.

  18. 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)

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

    Science.gov (United States)

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

    2018-01-29

    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 of methodologies have been adapted or developed over the last decades that allow minimal or non-invasive live-cell imaging in the context of tissues. Combined with the ease to generate transgenic reporter lines in specific genetic backgrounds or accessions, we are witnessing a blooming in plant cell biology. However, the imaging of plant cells entails a number of specific challenges, such as high levels of autofluorescence, light scattering that is caused by cell walls and their sensitivity to environmental conditions. Quantitative live-cell imaging in plants therefore requires adapting or developing imaging techniques, as well as mounting and incubation systems, such as micro-fluidics. Here, we discuss some of these obstacles, and review a number of selected state-of-the-art techniques, such as two-photon imaging, light sheet microscopy and variable angle epifluorescence microscopy that allow high performance and minimal invasive live-cell imaging in plants. © 2018. Published by The Company of Biologists Ltd.

  20. [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.

  1. clusters

    Indian Academy of Sciences (India)

    2017-09-27

    Sep 27, 2017 ... while CuCoNO, Co3NO, Cu3CoNO, Cu2Co3NO, Cu3Co3NO and Cu6CoNO clusters display stronger chemical stability. Magnetic and electronic properties are also discussed. The magnetic moment is affected by charge transfer and the spd hybridization. Keywords. CumConNO (m + n = 2–7) clusters; ...

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

    Science.gov (United States)

    Lahne, Manuela; Hyde, David R

    2017-08-01

    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.

  3. 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.

  4. PALM and STORM: unlocking live-cell super-resolution

    CSIR Research Space (South Africa)

    Henriques, R

    2011-05-01

    Full Text Available , hampering it from reaching its full potential for revealing the dynamic interactions in biology occurring at the single molecule level. Here we examine recent advances in the super-resolution imaging of living cells by reviewing recent breakthroughs...

  5. 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...

  6. 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.

  7. Super-resolution microscopy of living bacterial cells

    Science.gov (United States)

    Ponomareva, E. V.; Vishnyakov, I. E.; Morozova, N. E.; Polinovskaya, V. S.; Khodorkovskii, M. A.; Vedyaykin, A. D.

    2017-11-01

    Currently several methods of super-resolution optical microscopy are known. One of them – super-resolution radial fluctuations (SRRF) microscopy – was successfully used in present work to study the structures, formed by FtsZ protein in living E.coli cells with a resolution well below the diffraction limit.

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

    DEFF Research Database (Denmark)

    Liesche, Johannes; Schulz, Alexander

    2015-01-01

    cell wall interface. Transport through plasmodesmata, the cell wall channels that directly connect plant cells, is regulated not only by a fixed size exclusion limit, but also by physiological and pathological adaptation. The noninvasive approach described here offers the possibility of precisely...... 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...

  9. Emission spectra profiling of fluorescent proteins in living plant cells.

    Science.gov (United States)

    Mylle, Evelien; Codreanu, Mirela-Corina; Boruc, Joanna; Russinova, Eugenia

    2013-04-03

    Fluorescence imaging at high spectral resolution allows the simultaneous recording of multiple fluorophores without switching optical filters, which is especially useful for time-lapse analysis of living cells. The collected emission spectra can be used to distinguish fluorophores by a computation analysis called linear unmixing. The availability of accurate reference spectra for different fluorophores is crucial for this type of analysis. The reference spectra used by plant cell biologists are in most cases derived from the analysis of fluorescent proteins in solution or produced in animal cells, although these spectra are influenced by both the cellular environment and the components of the optical system. For instance, plant cells contain various autofluorescent compounds, such as cell wall polymers and chlorophyll, that affect the spectral detection of some fluorophores. Therefore, it is important to acquire both reference and experimental spectra under the same biological conditions and through the same imaging systems. Entry clones (pENTR) of fluorescent proteins (FPs) were constructed in order to create C- or N-terminal protein fusions with the MultiSite Gateway recombination technology. The emission spectra for eight FPs, fused C-terminally to the A- or B-type cyclin dependent kinases (CDKA;1 and CDKB1;1) and transiently expressed in epidermal cells of tobacco (Nicotiana benthamiana), were determined by using the Olympus FluoView™ FV1000 Confocal Laser Scanning Microscope. These experimental spectra were then used in unmixing experiments in order to separate the emission of fluorophores with overlapping spectral properties in living plant cells. Spectral imaging and linear unmixing have a great potential for efficient multicolor detection in living plant cells. The emission spectra for eight of the most commonly used FPs were obtained in epidermal cells of tobacco leaves and used in unmixing experiments. The generated set of FP Gateway entry vectors

  10. 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.).

  11. 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\\

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

    Science.gov (United States)

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

    2017-05-01

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

  13. Planar patch-clamp force microscopy on living cells

    Energy Technology Data Exchange (ETDEWEB)

    Pamir, Evren [Center for Nano Science, Ludwig-Maximilians University, Amalienstr 54, 80799 Munich (Germany); George, Michael; Fertig, Niels [Nanion Technologies GmbH, Erzgiessereistr. 4, 80335 Munich (Germany); Benoit, Martin [Center for Nano Science, Ludwig-Maximilians University, Amalienstr 54, 80799 Munich (Germany)], E-mail: martin.benoit@physik.uni-muenchen.de

    2008-05-15

    Here we report a new combination of the patch-clamp technique with the atomic force microscope (AFM). A planar patch-clamp chip microstructured from borosilicate glass was used as a support for mechanical probing of living cells. The setup not only allows for immobilizing even a non-adherent cell for measurements of its mechanical properties, but also for simultaneously measuring the electrophysiological properties of a single cell. As a proof of principle experiment we measured the voltage-induced membrane movement of HEK293 and Jurkat cells in the whole-cell voltage clamp configuration. The results of these measurements are in good agreement with previous studies. By using the planar patch-clamp chip for immobilization, the AFM not only can image non-adhering cells, but also gets easily access to an electrophysiologically controlled cellular probe at low vibrational noise.

  14. 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.

  15. Live-Cell High Content Screening in Drug Development.

    Science.gov (United States)

    Esner, Milan; Meyenhofer, Felix; Bickle, Marc

    2018-01-01

    In the past decade, automated microscopy has become an important tool for the drug discovery and development process. The establishment of imaging modalities as screening tools depended on technological breakthroughs in the domain of automated microscopy and automated image analysis. These types of assays are often referred to as high content screening or high content analysis (HCS/HCA). The driving force to adopt imaging for drug development is the quantity and quality of cellular information that can be collected and the enhanced physiological relevance of cellular screening compared to biochemical screening. Most imaging in drug development is performed on fixed cells as this allows uncoupling the preparation of the cells from the acquisition of the images. Live-cell imaging is technically challenging, but is very useful for many aspects of the drug development pipeline such as kinetic studies of compound mode of action or to analyze the motion of cellular components. Most vendors of HCS microscopy systems offer the option of environmental chambers and onboard pipetting on their platforms. This reflects the wish and desire of many customers to have the ability to perform live-cell assays on their HCS automated microscopes. This book chapter summarizes the challenges and advantages of live-cell imaging in drug discovery. Examples of applications are presented and the motivation to perform these assays in kinetic mode is discussed.

  16. 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...

  17. Distinct cell clusters touching islet cells induce islet cell replication in association with over-expression of Regenerating Gene (REG protein in fulminant type 1 diabetes.

    Directory of Open Access Journals (Sweden)

    Kaoru Aida

    Full Text Available BACKGROUND: Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs, extracellular matrix (ECM, and possible cell clusters, are unclear. PROCEDURES: The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans. RESULT: Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells. CONCLUSION: The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells.

  18. Comparative genomics of natural killer cell receptor gene clusters.

    Directory of Open Access Journals (Sweden)

    James Kelley

    2005-08-01

    Full Text Available Many receptors on natural killer (NK cells recognize major histocompatibility complex class I molecules in order to monitor unhealthy tissues, such as cells infected with viruses, and some tumors. Genes encoding families of NK receptors and related sequences are organized into two main clusters in humans: the natural killer complex on Chromosome 12p13.1, which encodes C-type lectin molecules, and the leukocyte receptor complex on Chromosome 19q13.4, which encodes immunoglobulin superfamily molecules. The composition of these gene clusters differs markedly between closely related species, providing evidence for rapid, lineage-specific expansions or contractions of sets of loci. The choice of NK receptor genes is polarized in the two species most studied, mouse and human. In mouse, the C-type lectin-related Ly49 gene family predominates. Conversely, the single Ly49 sequence is a pseudogene in humans, and the immunoglobulin superfamily KIR gene family is extensive. These different gene sets encode proteins that are comparable in function and genetic diversity, even though they have undergone species-specific expansions. Understanding the biological significance of this curious situation may be aided by studying which NK receptor genes are used in other vertebrates, especially in relation to species-specific differences in genes for major histocompatibility complex class I molecules.

  19. 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

  20. Clustered DNA damages induced in human hematopoietic cells by low doses of ionizing radiation

    Science.gov (United States)

    Sutherland, Betsy M.; Bennett, Paula V.; Cintron-Torres, Nela; Hada, Megumi; Trunk, John; Monteleone, Denise; Sutherland, John C.; Laval, Jacques; Stanislaus, Marisha; Gewirtz, Alan

    2002-01-01

    Ionizing radiation induces clusters of DNA damages--oxidized bases, abasic sites and strand breaks--on opposing strands within a few helical turns. Such damages have been postulated to be difficult to repair, as are double strand breaks (one type of cluster). We have shown that low doses of low and high linear energy transfer (LET) radiation induce such damage clusters in human cells. In human cells, DSB are about 30% of the total of complex damages, and the levels of DSBs and oxidized pyrimidine clusters are similar. The dose responses for cluster induction in cells can be described by a linear relationship, implying that even low doses of ionizing radiation can produce clustered damages. Studies are in progress to determine whether clusters can be produced by mechanisms other than ionizing radiation, as well as the levels of various cluster types formed by low and high LET radiation.

  1. cluster

    Indian Academy of Sciences (India)

    has been investigated electrochemically in positive and negative microenvironments, both in solution and in film. Charge nature around the active centre ... in plants, bacteria and also in mammals. This cluster is also an important constituent of a ..... selection of non-cysteine amino acid in the active centre of Rieske proteins.

  2. Glioblastoma stem cell differentiation into endothelial cells evidenced through live-cell imaging.

    Science.gov (United States)

    Mei, Xin; Chen, Yin-Sheng; Chen, Fu-Rong; Xi, Shao-Yan; Chen, Zhong-Ping

    2017-08-01

    Glioblastoma cell-initiated vascularization is an alternative angiogenesis called vasculogenic mimicry. However, current knowledge on the mechanism of de novo vessel formation from glioblastoma stem cells (GSCs) is limited. Sixty-four glioblastoma samples from patients and 10 fluorescent glioma xenograft samples were examined by immunofluorescence staining for endothelial marker (CD34 and CD31) and glial cell marker (glial fibrillary acidic protein [GFAP]) expression. GSCs were then isolated from human glioblastoma tissue and CD133+/Sox2+ red fluorescent protein-containing (RFP)-GSC-1 cells were established. The ability of these cells to form vascular structures was examined by live-cell imaging of 3D cultures. CD34-GFAP or CD31-GFAP coexpressing glioblastoma-derived endothelial cells (GDEC) were found in 30 of 64 (46.9%) of clinical glioblastoma samples. In those 30 samples, GDEC were found to form vessel structures in 21 (70%) samples. Among 21 samples with GDEC vessels, the CD34+ GDEC vessels and CD31+ GDEC vessels accounted for about 14.16% and 18.08% of total vessels, respectively. In the xenograft samples, CD34+ GDEC were found in 7 out of 10 mice, and 4 out of 7 mice had CD34+ GDEC vessels. CD31+ GDEC were also found in 7 mice, and 4 mice had CD31+ GDEC vessels (10 mice in total). Through live-cell imaging, we observed gradual CD34 expression when cultured with vascular endothelial growth factor in some glioma cells, and a dynamic increase in endothelial marker expression in RFP-GSC-1 in vitro was recorded. Cells expressed CD34 (9.46%) after 6 hours in culture. The results demonstrated that GSCs may differentiate into endothelial cells and promote angiogenesis in glioblastomas. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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

    Science.gov (United States)

    Dreissig, Steven; Schiml, Simon; Schindele, Patrick; Weiss, Oda; Rutten, Twan; Schubert, Veit; Gladilin, Evgeny; Mette, Michael F; Puchta, Holger; Houben, Andreas

    2017-08-01

    Elucidating the spatiotemporal organization of the genome inside the nucleus is imperative to our understanding of 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 clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9). By fusing eGFP/mRuby2 to catalytically inactive versions 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 over 30 min 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 the 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. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

  4. Live imaging of adult neural stem cells in rodents

    Directory of Open Access Journals (Sweden)

    Felipe eOrtega

    2016-03-01

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

  5. Live Imaging of Adult Neural Stem Cells in Rodents

    Science.gov (United States)

    Ortega, Felipe; Costa, Marcos R.

    2016-01-01

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

  6. Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5-chloromethylfluorescein diacetate.

    Science.gov (United States)

    MacIntyre, Hugh L; Cullen, John J

    2016-08-01

    Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5-chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat-killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per-cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live-dead classification was essentially error free. But overlap between the frequency distributions of living and heat-killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat-killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8-10 of 24 species (i.e., 33%-42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone. © 2016 The Authors. Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America.

  7. 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.

  8. Lethal action of thermal neutron to living cells

    International Nuclear Information System (INIS)

    Kawai, Kenichi; Akahoshi, Mitsuhiko

    1975-01-01

    Using Amoeba Proteus (DNA content was 5x10 -10 g/cell), action mechanism of thermal neutron was described by using death of cells as an index. Using mononuclear type (M-type) and polynuclear type (P-type) of Amoeba, investigation was made as to whether the number of nucleus contained in cell was related to death of cell by radiation. As compared to gamma-ray, marked difference between sensitivity of both types of Amoeba was not recognized. It was suggested that nucleus was important as a place of death of cell by thermal neutron. In order to demonstrate that reaction of 31 P(n, γ) 32 Pwas a main cause of lethality, the following experiment was carried out. 1) In order to investigate that radical reaction was not a main cause of lethal action by thermal neutron, increase and decrease of radical reaction were observed by adding AET, Cysteine and BudR. As to thermal neutron action, effect of its chemical factors did not appeared. 2) In order to demonstrate that nuclear reaction of constituent atom of living body played an important part in death of cell, increase and decrease of nuclear reaction within cell were observed by taking Na 2 , B 4 and O 7 into Amoeba. It was suggested that nuclear reaction of cell constituent atom itself was important in death of cell. 3) Potentiation effect of gamma-ray and thermal neutron was investigated and its mechanism was discussed. (K. Serizawa)

  9. 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.

  10. Collective Dynamics of Intracellular Water in Living Cells

    Science.gov (United States)

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

    2012-02-01

    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.

  11. 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.

  12. 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

  13. Apical trafficking in epithelial cells: signals, clusters and motors.

    Science.gov (United States)

    Weisz, Ora A; Rodriguez-Boulan, Enrique

    2009-12-01

    In the early days of epithelial cell biology, researchers working with kidney and/or intestinal epithelial cell lines and with hepatocytes described the biosynthetic and recycling routes followed by apical and basolateral plasma membrane (PM) proteins. They identified the trans-Golgi network and recycling endosomes as the compartments that carried out apical-basolateral sorting. They described complex apical sorting signals that promoted association with lipid rafts, and simpler basolateral sorting signals resembling clathrin-coated-pit endocytic motifs. They also noticed that different epithelial cell types routed their apical PM proteins very differently, using either a vectorial (direct) route or a transcytotic (indirect) route. Although these original observations have generally held up, recent studies have revealed interesting complexities in the routes taken by apically destined proteins and have extended our understanding of the machinery required to sustain these elaborate sorting pathways. Here, we critically review the current status of apical trafficking mechanisms and discuss a model in which clustering is required to recruit apical trafficking machineries. Uncovering the mechanisms responsible for polarized trafficking and their epithelial-specific variations will help understand how epithelial functional diversity is generated and the pathogenesis of many human diseases.

  14. Long-lived charge-separated states in ligand-stabilized silver clusters

    KAUST Repository

    Pelton, Matthew

    2012-07-25

    Recently developed synthesis methods allow for the production of atomically monodisperse clusters of silver atoms stabilized in solution by aromatic thiol ligands, which exhibit intense absorption peaks throughout the visible and near-IR spectral regions. Here we investigated the time-dependent optical properties of these clusters. We observed two kinetic processes following ultrafast laser excitation of any of the absorption peaks: a rapid decay, with a time constant of 1 ps or less, and a slow decay, with a time constant that can be longer than 300 ns. Both time constants decrease as the polarity of the solvent increases, indicating that the two processes correspond to the formation and recombination, respectively, of a charge-separated state. The long lifetime of this state and the broad optical absorption spectrum mean that the ligand-stabilized silver clusters are promising materials for solar energy harvesting. © 2012 American Chemical Society.

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

    NARCIS (Netherlands)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

  17. Characterization of exogenous DNA mobility in live cells through fluctuation correlation spectroscopy

    Science.gov (United States)

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

    2015-01-01

    The spatial-temporal dynamics of delivered DNA is a critical aspect influencing successful gene delivery. A comprehensive model of DNA lipoplex trafficking through live cells has yet to be demonstrated. Here the bioimaging approaches Raster Image Correlation Spectroscopy (RICS) and image-Means Square Displacement (iMSD) were applied to quantify DNA mechanical dynamics in live cells. DNA lipoplexes formed from DNA with a range of 21 bp to 5.5 kbp exhibited a similar range of motion within the cytoplasm of myoblast cells regardless of size. However, the rate of motion was dictated by the intracellular location, and DNA cluster size. This analysis demonstrated that the different transport mechanisms either had a size dependent mobility, including random diffusion, whereas other mechanisms were not influenced by the DNA size such as active transport. The transport mechanisms identified followed a spatial dependence comparable to viral trafficking of non-active transport mechanism upon cellular entry, active transport within the cytoplasm and further inactive transportation along the peri-nuclear region. This study provides the first real-time insight into the trafficking of DNA delivered through lipofection using image-based fluctuation correlation spectroscopy approaches. Thereby, gaining information with single particle sensitivity to develop a deeper understanding of DNA lipoplex delivery through the cell. PMID:26354725

  18. Rotational magnetic endosome microrheology: Viscoelastic architecture inside living cells

    Science.gov (United States)

    Wilhelm, C.; Gazeau, F.; Bacri, J.-C.

    2003-06-01

    The previously developed technique of magnetic rotational microrheology [Phys. Rev. E 67, 011504 (2003)] is proposed to investigate the rheological properties of the cell interior. An endogeneous magnetic probe is obtained inside living cells by labeling intracellular compartments with magnetic nanoparticles, following the endocytosis mechanism, the most general pathway used by eucaryotic cells to internalize substances from an extracellular medium. Primarily adsorbed on the plasma membrane, the magnetic nanoparticles are first internalized within submicronic membrane vesicles (100 nm diameter) to finally concentrate inside endocytotic intracellular compartments (0.6 μm diameter). These magnetic endosomes attract each other and form chains within the living cell when submitted to an external magnetic field. Here we demonstrate that these chains of magnetic endosomes are valuable tools to probe the intracellular dynamics at very local scales. The viscoelasticity of the chain microenvironment is quantified in terms of a viscosity η and a relaxation time τ by analyzing the rotational dynamics of each tested chain in response to a rotation of the external magnetic field. The viscosity η governs the long time flow of the medium surrounding the chains and the relaxation time τ reflects the proportion of solidlike versus liquidlike behavior (τ=η/G, where G is the high-frequency shear modulus). Measurements in HeLa cells show that the cell interior is a highly heterogeneous structure, with regions where chains are embedded inside a dense viscoelastic matrix and other domains where chains are surrounded by a less rigid viscoelastic material. When one compound of the cell cytoskeleton is disrupted (microfilaments or microtubules), the intracellular viscoelasticity becomes less heterogeneous and more fluidlike, in the sense of both a lower viscosity and a lower relaxation time.

  19. Vibrio cholerae biofilm growth program and architecture revealed by single-cell live imaging.

    Science.gov (United States)

    Yan, Jing; Sharo, Andrew G; Stone, Howard A; Wingreen, Ned S; Bassler, Bonnie L

    2016-09-06

    Biofilms are surface-associated bacterial communities that are crucial in nature and during infection. Despite extensive work to identify biofilm components and to discover how they are regulated, little is known about biofilm structure at the level of individual cells. Here, we use state-of-the-art microscopy techniques to enable live single-cell resolution imaging of a Vibrio cholerae biofilm as it develops from one single founder cell to a mature biofilm of 10,000 cells, and to discover the forces underpinning the architectural evolution. Mutagenesis, matrix labeling, and simulations demonstrate that surface adhesion-mediated compression causes V. cholerae biofilms to transition from a 2D branched morphology to a dense, ordered 3D cluster. We discover that directional proliferation of rod-shaped bacteria plays a dominant role in shaping the biofilm architecture in V. cholerae biofilms, and this growth pattern is controlled by a single gene, rbmA Competition analyses reveal that the dense growth mode has the advantage of providing the biofilm with superior mechanical properties. Our single-cell technology can broadly link genes to biofilm fine structure and provides a route to assessing cell-to-cell heterogeneity in response to external stimuli.

  20. Clusters in social behaviour of female domestic cats (Felis silvestris catus) living in confinement

    NARCIS (Netherlands)

    vandenBos, R; de Vries, Han

    1996-01-01

    Associations between different agonistic and affiliative behavioural patterns of female domestic cats (Felis silvestris catus) were studied. In three groups of intact cats living in confinement frequencies of fourteen agonistic and affiliative behavioural patterns were recorded. The technique of

  1. 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...... scanning Raman microscope (Alpha300R) from Witec and sub-μm spatially resolved Raman images were obtained using a 532 nm excitation wavelength....

  2. Detecting protein-protein interactions in living cells

    DEFF Research Database (Denmark)

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

    2009-01-01

    -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...... 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...

  3. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging.

    NARCIS (Netherlands)

    Hoebe, R.A.; van Oven, C.H.; Gadella, Th.W.J.; Dhonukshe, P.B.; van Noorden, C.J.F.; Manders, E.M.M.

    2007-01-01

    Fluorescence microscopy of living cells enables visualization of the dynamics and interactions of intracellular molecules. However, fluorescence live-cell imaging is limited by photobleaching and phototoxicity induced by the excitation light. Here we describe controlled light-exposure microscopy

  4. Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging

    NARCIS (Netherlands)

    Hoebe, R. A.; van Oven, C. H.; Gadella, T. W. J.; Dhonukshe, P. B.; van Noorden, C. J. F.; Manders, E. M. M.

    2007-01-01

    Fluorescence microscopy of living cells enables visualization of the dynamics and interactions of intracellular molecules. However, fluorescence live-cell imaging is limited by photobleaching and phototoxicity induced by the excitation light. Here we describe controlled light-exposure microscopy (

  5. 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.

  6. 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.

  7. 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.

  8. 3-dimensional forces and molecular dynamics of live cells

    Science.gov (United States)

    Hur, Sung Sik; Li, Yi-Shuan; Park, Joon Seok; Hu, Ying-Li; Chien, Shu

    2010-08-01

    The forces exerted by an adherent cell on a substrate were studied previously only in the two-dimensions (2D) tangential to the substrate surface. We used a novel technique to measure the three-dimensional (3D) stresses exerted by live bovine aortic endothelial cells (BAECs) on polyacrylamide deformable substrate, with particular emphasis on the 3D forces of focal adhesions. On 3D images acquired by confocal microscopy, displacements were determined with imageprocessing programs, and stresses in tangential (XY) and normal (Z) directions were computed by finite element method (FEM). BAECs generated stress in normal direction (Tz) with an order of magnitude comparable to that in tangential direction (Txy). Tz is upward at the cell edge and downward under the nucleus, changing continuously with a sign reversal between cell edge and nucleus edge. With the use of green fluorescent protein (GFP) labeled paxillin, the dynamics of this intracellular molecule were studied concurrently with the measurement of 3D forces. In the dynamic region, including the new lamellapodium forming region in the front and the retracting region in the rear, the tangential forces (Fxy) are correlated with the size of the focal adhesions (FAs) much more strongly than those in the stable region under the nucleus. In the dynamic region, normal force (Fz) was upward and positively correlated with FA size, while Fz in the stable region was downward and negatively correlated with FA size. These findings show the influence of the size of FAs on the 3D forces they exert on the substrate. This technique can be applied to study any adherent type of live cells to assess their biomechanical dynamics in conjunction with biochemical and functional activities, thus elucidating cellular functions in health and disease.

  9. 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.

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

    Science.gov (United States)

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

    2017-06-02

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

  11. 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...

  12. 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...

  13. 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

  14. 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.

  15. Versatile optical manipulation system for inspection, laser processing, and isolation of individual living cells

    Science.gov (United States)

    Stuhrmann, B.; Jahnke, H.-G.; Schmidt, M.; Jähn, K.; Betz, T.; Müller, K.; Rothermel, A.; Käs, J.; Robitzki, A. A.

    2006-06-01

    Isolation of individual cells from a heterogeneous cell population is an invaluable step in the analysis of single cell properties. The demands in molecular and cellular biology as well as molecular medicine are the selection, isolation, and monitoring of single cells and cell clusters of biopsy material. Of particular interest are methods which complement a passive optical or spectroscopic selection with a variety of active single cell processing techniques such as mechanical, biochemical, or genetic manipulation prior to isolation. Sophisticated laser-based cell processing systems are available which can perform single cell processing in a contact-free and sterile manner. Until now, however, these multipurpose turnkey systems offer only basic micromanipulation and are not easily modified or upgraded, whereas laboratory situations often demand simple but versatile and adaptable solutions. We built a flexible laser micromanipulation platform combining contact-free microdissection and catapulting capabilities using a pulsed ultraviolet (337nm) laser with simultaneous generation of optical tweezing forces using a continuous wave infrared (1064nm) laser. The potential of our platform is exemplified with techniques such as local laser-induced injection of biomolecules into individual living cells, laser surgery, isolation of single cells by laser catapulting, and control of neuronal growth using optical gradient forces. Arbitrary dynamic optical force patterns can be created by fast laser scanning with acousto-optical deflectors and galvanometer mirrors, allowing multibeam contact-free micromanipulation, a prerequisite for reliable handling of material in laboratory-on-a-chip applications. All common microscopy techniques can be used simultaneously with the offered palette of micromanipulation methods. Taken together, we show that advanced optical micromanipulation systems can be designed which combine quality, cost efficiency, and adaptability.

  16. Live-cell Imaging Approaches for the Investigation of ...

    Science.gov (United States)

    BACKGROUND: Oxidant stress is arguably a universal feature in toxicology. Research studies on the role of oxidant stress induced by xenobiotic exposures have typically relied on the identification of damaged biomolecules using a variety of conventional biochemical and molecular techniques. However, there is increasing evidence that low-level exposure to a variety of toxicants dysregulates cellular physiology by interfering with redox-dependent processes.SCOPE OF REVIEW: The study of events involved in redox toxicology requires methodology capable of detecting transient modifications at relatively low signal strength. This article reviews the advantages of live-cell imaging for redox toxicology studies.MAJOR CONCLUSIONS: Toxicological studies with xenobiotics of supra-physiological reactivity require careful consideration when using fluorogenic sensors in order to avoid potential artifacts and false negatives. Fortunately, experiments conducted for the purpose of validating the use of these sensors in toxicological applications often yield unexpected insights into the mechanisms through which xenobiotic exposure induces oxidant stress.GENERAL SIGNIFICANCE: Live-cell imaging using a new generation of small molecule and genetically encoded fluorophores with excellent sensitivity and specificity affords unprecedented spatiotemporal resolution that is optimal for redox toxicology studies. This article is part of a Special Issue entitled Air Pollution, edited by Wenju

  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. 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.

  19. 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

  20. Measurement of GPCR-G protein activity in living cells.

    Science.gov (United States)

    Ratnayake, Kasun; Kankanamge, Dinesh; Senarath, Kanishka; Siripurapu, Praneeth; Weis, Nicole; Tennakoon, Mithila; Payton, John L; Karunarathne, Ajith

    2017-01-01

    G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors in eukaryotic genomes. They control a variety of cellular and physiological processes such as hormone secretion and heart rate, and therefore are associated with a majority of pathological conditions including cancer and heart diseases. Currently established assays to measure ligand-induced activation of GPCRs and G proteins possess limitations such as being time consuming, indirect, and expensive. Thus, an efficient method to measure GPCR-G protein activation is required to identify novel pharmacological modulators to control them and gain insights about molecular underpinnings of the associated pathways. Activation of GPCRs induces dissociation of G protein heterotrimers to form GαGTP and free Gβγ. Free Gβγ subunits have been shown to translocate reversibly from the plasma membrane to internal membranes. Gβγ translocation therefore represents the GPCR-G protein activation, and thus, imaging of this process can be used to quantify the kinetics and magnitude of the pathway activation-deactivation in real time in living cells. The objective of this chapter is to elaborate the protocols of (i) generation and optimization of the required sensor constructs; (ii) development of cell culture, transient transfection, imaging, and optogenetic procedures; (iii) imaging and data analysis methods; and (iv) stable cell line generation, pertaining to this assay to measure GPCR-G protein activation. © 2017 Elsevier Inc. All rights reserved.

  1. 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

  2. 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.

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

    Science.gov (United States)

    Sun, Sean

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

  4. Investigation of a cluster of syphilis, gonorrhea, and chlamydia cases among heterosexual Micronesians living on Oahu.

    Science.gov (United States)

    Katz, Alan R; Cadorna, Adrianne M; Lee, Maria Veneranda C; Komeya, Alan; Kiaha, Mandy; Ohye, Roy G

    2009-10-01

    A recent case investigation of secondary syphilis in a 27-year-old heterosexual Micronesian male and his 19-year-old chlamydia and syphilis co-infected female partner, conducted by the Hawaii State Department of Health, June to August 2007, identified a cluster of 13 case-patients with undiagnosed syphilis, chlamydia, and gonorrhea. The social network of Micronesians uncovered was characterized by having transient accommodations but a central gathering place. The critical factor in gaining access to this network was the establishment of a trusting relationship with a key social network member. Field interviews and the application of field diagnostic techniques helped to identify case-patients who otherwise would not have presented to a traditional office or clinic setting. Micronesians in Hawaii represent an at-risk population for adverse health indices including sexually transmitted diseases, based on their socioeconomic status.

  5. FLOCK cluster analysis of mast cell event clustering by high-sensitivity flow cytometry predicts systemic mastocytosis.

    Science.gov (United States)

    Dorfman, David M; LaPlante, Charlotte D; Pozdnyakova, Olga; Li, Betty

    2015-11-01

    In our high-sensitivity flow cytometric approach for systemic mastocytosis (SM), we identified mast cell event clustering as a new diagnostic criterion for the disease. To objectively characterize mast cell gated event distributions, we performed cluster analysis using FLOCK, a computational approach to identify cell subsets in multidimensional flow cytometry data in an unbiased, automated fashion. FLOCK identified discrete mast cell populations in most cases of SM (56/75 [75%]) but only a minority of non-SM cases (17/124 [14%]). FLOCK-identified mast cell populations accounted for 2.46% of total cells on average in SM cases and 0.09% of total cells on average in non-SM cases (P < .0001) and were predictive of SM, with a sensitivity of 75%, a specificity of 86%, a positive predictive value of 76%, and a negative predictive value of 85%. FLOCK analysis provides useful diagnostic information for evaluating patients with suspected SM, and may be useful for the analysis of other hematopoietic neoplasms. Copyright© by the American Society for Clinical Pathology.

  6. Half-lives for proton emission, alpha decay, cluster radioactivity, and cold fission processes calculated in a unified theoretical framework

    International Nuclear Information System (INIS)

    Duarte, S.B.; Tavares, O.A.P.; Guzman, F.; Dimarco, A.; Garcia, F.; Goncalves, M.

    2002-01-01

    Half-life values of spontaneous nuclear decay processes are presented in the framework of the Effective Liquid Drop Model (ELDM) using the combination of varying mass asymmetry shape description for the mass transfer with Werner-Wheeler's inertia coefficient V MAS /WW. The calculated half-lives of ground-state to ground-state transitions for the proton emission, alpha decay, cluster radioactivity, and cold fission processes are compared with experimental data. Results have shown that the ELDM is a very efficient model to describe these different decay processes in a same, unified theoretical framework. A Table listing the predicted half-life values, τ c is presented for all possible cases of spontaneous nuclear break-up such that -7.30 10 τ c [S] 10 (τ/τ c ) > -17.0, where τ is the total half-life of the parent nucleus. (author)

  7. Silicon nitride waveguide platform for fluorescence microscopy of living cells.

    Science.gov (United States)

    Tinguely, Jean-Claude; Helle, Øystein Ivar; Ahluwalia, Balpreet Singh

    2017-10-30

    Waveguide chip-based microscopy reduces the complexity of total internal reflection fluorescence (TIRF) microscopy, and adds features like large field of view illumination, decoupling of illumination and collection path and easy multimodal imaging. However, for the technique to become widespread there is a need of low-loss and affordable waveguides made of high-refractive index material. Here, we develop and report a low-loss silicon nitride (Si 3 N 4 ) waveguide platform for multi-color TIRF microscopy. Single mode conditions at visible wavelengths (488-660 nm) were achieved using shallow rib geometry. To generate uniform excitation over appropriate dimensions waveguide bends were used to filter-out higher modes followed by adiabatic tapering. Si 3 N 4 material is finally shown to be biocompatible for growing and imaging living cells.

  8. Optimized Tetrazine Derivatives for Rapid Bioorthogonal Decaging in Living Cells.

    Science.gov (United States)

    Fan, Xinyuan; Ge, Yun; Lin, Feng; Yang, Yi; Zhang, Gong; Ngai, William Shu Ching; Lin, Zhi; Zheng, Siqi; Wang, Jie; Zhao, Jingyi; Li, Jie; Chen, Peng R

    2016-11-02

    The inverse-electron-demand Diels-Alder (iDA) reaction has recently been repurposed as a bioorthogonal decaging reaction by accelerating the elimination process after an initial cycloaddition between trans-cyclooctene (TCO) and tetrazine (TZ). Herein, we systematically surveyed 3,6-substituted TZ derivatives by using a fluorogenic TCO-coumarin reporter followed by LC-MS analysis, which revealed that the initial iDA cycloaddition step was greatly accelerated by electron-withdrawing groups (EWGs) while the subsequent elimination step was strongly suppressed by EWGs. In addition, smaller substituents facilitated the decaging process. These findings promoted us to design and test unsymmetric TZs bearing an EWG group and a small non-EWG group at the 3- and 6-position, respectively. These TZs showed remarkably enhanced decaging rates, enabling rapid iDA-mediated protein activation in living cells. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

    Relich, Peter Kristopher, II

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

  10. Segmentation of the Clustered Cells with Optimized Boundary Detection in Negative Phase Contrast Images.

    Directory of Open Access Journals (Sweden)

    Yuliang Wang

    Full Text Available Cell image segmentation plays a central role in numerous biology studies and clinical applications. As a result, the development of cell image segmentation algorithms with high robustness and accuracy is attracting more and more attention. In this study, an automated cell image segmentation algorithm is developed to get improved cell image segmentation with respect to cell boundary detection and segmentation of the clustered cells for all cells in the field of view in negative phase contrast images. A new method which combines the thresholding method and edge based active contour method was proposed to optimize cell boundary detection. In order to segment clustered cells, the geographic peaks of cell light intensity were utilized to detect numbers and locations of the clustered cells. In this paper, the working principles of the algorithms are described. The influence of parameters in cell boundary detection and the selection of the threshold value on the final segmentation results are investigated. At last, the proposed algorithm is applied to the negative phase contrast images from different experiments. The performance of the proposed method is evaluated. Results show that the proposed method can achieve optimized cell boundary detection and highly accurate segmentation for clustered cells.

  11. Segmentation of the Clustered Cells with Optimized Boundary Detection in Negative Phase Contrast Images.

    Science.gov (United States)

    Wang, Yuliang; Zhang, Zaicheng; Wang, Huimin; Bi, Shusheng

    2015-01-01

    Cell image segmentation plays a central role in numerous biology studies and clinical applications. As a result, the development of cell image segmentation algorithms with high robustness and accuracy is attracting more and more attention. In this study, an automated cell image segmentation algorithm is developed to get improved cell image segmentation with respect to cell boundary detection and segmentation of the clustered cells for all cells in the field of view in negative phase contrast images. A new method which combines the thresholding method and edge based active contour method was proposed to optimize cell boundary detection. In order to segment clustered cells, the geographic peaks of cell light intensity were utilized to detect numbers and locations of the clustered cells. In this paper, the working principles of the algorithms are described. The influence of parameters in cell boundary detection and the selection of the threshold value on the final segmentation results are investigated. At last, the proposed algorithm is applied to the negative phase contrast images from different experiments. The performance of the proposed method is evaluated. Results show that the proposed method can achieve optimized cell boundary detection and highly accurate segmentation for clustered cells.

  12. Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.

    Science.gov (United States)

    Shuai, Hongyan; Xu, Yunjian; Yu, Qian; Gylfe, Erik; Tengholm, Anders

    2016-10-01

    The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.

  13. 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)

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

    Science.gov (United States)

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

    2015-09-01

    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. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  15. 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.

  16. 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

  17. Reasoning Exercises in Assisted Living: a cluster randomized trial to improve reasoning and everyday problem solving

    Science.gov (United States)

    Williams, Kristine; Herman, Ruth; Bontempo, Daniel

    2014-01-01

    Purpose of the study Assisted living (AL) residents are at risk for cognitive and functional declines that eventually reduce their ability to care for themselves, thereby triggering nursing home placement. In developing a method to slow this decline, the efficacy of Reasoning Exercises in Assisted Living (REAL), a cognitive training intervention that teaches everyday reasoning and problem-solving skills to AL residents, was tested. Design and methods At thirteen randomized Midwestern facilities, AL residents whose Mini Mental State Examination scores ranged from 19–29 either were trained in REAL or a vitamin education attention control program or received no treatment at all. For 3 weeks, treated groups received personal training in their respective programs. Results Scores on the Every Day Problems Test for Cognitively Challenged Elders (EPCCE) and on the Direct Assessment of Functional Status (DAFS) showed significant increases only for the REAL group. For EPCCE, change from baseline immediately postintervention was +3.10 (P<0.01), and there was significant retention at the 3-month follow-up (d=2.71; P<0.01). For DAFS, change from baseline immediately postintervention was +3.52 (P<0.001), although retention was not as strong. Neither the attention nor the no-treatment control groups had significant gains immediately postintervention or at follow-up assessments. Post hoc across-group comparison of baseline change also highlights the benefits of REAL training. For EPCCE, the magnitude of gain was significantly larger in the REAL group versus the no-treatment control group immediately postintervention (d=3.82; P<0.01) and at the 3-month follow-up (d=3.80; P<0.01). For DAFS, gain magnitude immediately postintervention for REAL was significantly greater compared with in the attention control group (d=4.73; P<0.01). Implications REAL improves skills in everyday problem solving, which may allow AL residents to maintain self-care and extend AL residency. This benefit

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

    Science.gov (United States)

    Van Valen, David A.; Lane, Keara M.; Quach, Nicolas T.; Maayan, Inbal

    2016-01-01

    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. PMID:27814364

  19. 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

  20. T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters

    Energy Technology Data Exchange (ETDEWEB)

    Manz, Boryana N. [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Univ. of California, Berkeley, CA (United States); Jackson, Bryan L. [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Petit, Rebecca S. [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Dustin, Michael L. [New York School of Medicine, New York, NY (United States); Groves, Jay [Howard Hughes Medical Inst., Chevy Chase, MD (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)

    2011-05-31

    T cells react to extremely small numbers of activating agonist peptides. Spatial organization of T-cell receptors (TCR) and their peptide-major histocompatibility complex (pMHC) ligands into microclusters is correlated with T-cell activation. In this study, we have designed an experimental strategy that enables control over the number of agonist peptides per TCR cluster, without altering the total number engaged by the cell. Supported membranes, partitioned with grids of barriers to lateral mobility, provide an effective way of limiting the total number of pMHC ligands that may be assembled within a single TCR cluster. Observations directly reveal that restriction of pMHC content within individual TCR clusters can decrease T-cell sensitivity for triggering initial calcium flux at fixed total pMHC density. Further analysis suggests that triggering thresholds are determined by the number of activating ligands available to individual TCR clusters, not by the total number encountered by the cell. Results from a series of experiments in which the overall agonist density and the maximum number of agonist per TCR cluster are independently varied in primary T cells indicate that the most probable minimal triggering unit for calcium signaling is at least four pMHC in a single cluster for this system. In conclusion, this threshold is unchanged by inclusion of coagonist pMHC, but costimulation of CD28 by CD80 can modulate the threshold lower.

  1. Correlating cell architecture with osteogenesis: first steps towards live single cell monitoring.

    Science.gov (United States)

    Born, A-K; Rottmar, M; Lischer, S; Pleskova, M; Bruinink, A; Maniura-Weber, K

    2009-10-23

    Cell shape and regulation of biological processes such as proliferation and differentiation are to a large degree connected. Investigation of the possible relationship between cell shape and function is therefore important for developing new material concepts for medical applications as well as developing novel cell based sensors. Cell spreading requires a firm contact with the underlying substrate, with focal contacts (FC) being the primary sites of adhesion. They consist of a large number of clustered transmembrane proteins (integrins). FC integrins connect the cell cytoskeleton with the cell substratum. It has been demonstrated that cell spreading increases osteoblast differentiation in pre-osteoblastic progenitors. The gradual process of osteogenesis can be followed by different proteins being expressed at various time points, comprising early (e.g., bone-specific alkaline phosphatase (bALP)) and late genes (e.g., osteocalcin (OC)). In the present study we have used immunohistochemistry and RT-PCR to determine osteogenic differentiation of human bone cells (HBC). For online monitoring, fluorescently-tagged actin and vinculin were used for transfection of HBCs. Transfection of HBCs with an OC promoter gene construct allowed us to online monitor the gradual process of osteogenesis. We found distinct changes in cell architecture upon osteogenic differentiation thus providing evidence for the connection between cell shape and functional state.

  2. Correlating cell architecture with osteogenesis: first steps towards live single cell monitoring

    Directory of Open Access Journals (Sweden)

    A-K Born

    2009-10-01

    Full Text Available Cell shape and regulation of biological processes such as proliferation and differentiation are to a large degree connected. Investigation of the possible relationship between cell shape and function is therefore important for developing new material concepts for medical applications as well as developing novel cell based sensors. Cell spreading requires a firm contact with the underlying substrate, with focal contacts (FC being the primary sites of adhesion. They consist of a large number of clustered transmembrane proteins (integrins. FC integrins connect the cell cytoskeleton with the cell substratum. It has been demonstrated that cell spreading increases osteoblast differentiation in pre-osteoblastic progenitors. The gradual process of osteogenesis can be followed by different proteins being expressed at various time points, comprising early (e.g., bone-specific alkaline phosphatase (bALP and late genes (e.g., osteocalcin (OC. In the present study we have used immunohistochemistry and RT-PCR to determine osteogenic differentiation of human bone cells (HBC. For online monitoring, fluorescently-tagged actin and vinculin were used for transfection of HBCs. Transfection of HBCs with an OC promoter gene construct allowed us to online monitor the gradual process of osteogenesis. We found distinct changes in cell architecture upon osteogenic differentiation thus providing evidence for the connection between cell shape and functional state.

  3. 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)

  4. Processing of radiation-induced clustered DNA damage generates DSB in mammalian cells

    International Nuclear Information System (INIS)

    Gulston, M.K.; De Lara, C.M.; Davis, E.L.; Jenner, T.J.; O'Neill, P.

    2003-01-01

    Full text: Clustered DNA damage sites, in which two or more lesions are formed within a few helical turns of the DNA after passage of a single radiation track, are signatures of DNA modifications induced by ionizing radiation in mammalian cell. With 60 Co-radiation, the abundance of clustered DNA damage induced in CHO cells is ∼4x that of prompt double strand breaks (DSB) determined by PFGE. Less is known about the processing of non-DSB clustered DNA damage induced in cells. To optimize observation of any additional DSB formed during processing of DNA damage at 37 deg C, xrs-5 cells deficient in non-homologous end joining were used. Surprisingly, ∼30% of the DSB induced by irradiation at 37 deg C are rejoined within 4 minutes in both mutant and wild type cells. No significant mis-repair of these apparent DSB was observed. It is suggested that a class of non-DSB clustered DNA damage is formed which repair correctly within 4 min but, if 'trapped' prior to repair, are converted into DSB during the lysis procedure of PFGE. However at longer times, a proportion of non-DSB clustered DNA damage sites induced by γ-radiation are converted into DSB within ∼30 min following post-irradiation incubation at 37 deg C. The corresponding formation of additional DSB was not apparent in wild type CHO cells. From these observations, it is estimated that only ∼10% of the total yield of non DSB clustered DNA damage sites are converted into DSB through cellular processing. The biological consequences that the majority of non-DSB clustered DNA damage sites are not converted into DSBs may be significant even at low doses, since a finite chance exists of these clusters being formed in a cell by a single radiation track

  5. 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.

  6. Clustered Ergot Alkaloids Modulate Cell-mediated Cytotoxicity

    Czech Academy of Sciences Publication Activity Database

    Křen, Vladimír; Fišerová, Anna; Weignerová, Lenka; Stibor, I.; Halada, Petr; Přikrylová, Věra; Sedmera, Petr; Pospíšil, Miloslav

    2002-01-01

    Roč. 10, - (2002), s. 415-424 ISSN 0968-0896 R&D Projects: GA AV ČR IAA4020901; GA ČR GA310/98/0347 Institutional research plan: CEZ:AV0Z5020903 Keywords : clustered * ergot * alkaloids Subject RIV: EE - Microbiology, Virology Impact factor: 2.043, year: 2002

  7. 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

  8. Nonspecific CD8+T Cells and Dendritic Cells/Macrophages Participate in Formation of CD8+T Cell-Mediated Clusters against Malaria Liver-Stage Infection.

    Science.gov (United States)

    Akbari, Masoud; Kimura, Kazumi; Bayarsaikhan, Ganchimeg; Kimura, Daisuke; Miyakoda, Mana; Juriasingani, Smriti; Yuda, Masao; Amino, Rogerio; Yui, Katsuyuki

    2018-04-01

    CD8 + T cells are the major effector cells that protect against malaria liver-stage infection, forming clusters around Plasmodium -infected hepatocytes and eliminating parasites after a prolonged interaction with these hepatocytes. We aimed to investigate the roles of specific and nonspecific CD8 + T cells in cluster formation and protective immunity. To this end, we used Plasmodium berghei ANKA expressing ovalbumin as well as CD8 + T cells from transgenic mice expressing a T cell receptor specific for ovalbumin (OT-I) and CD8 + T cells specific for an unrelated antigen, respectively. While antigen-specific CD8 + T cells were essential for cluster formation, both antigen-specific and nonspecific CD8 + T cells joined the clusters. However, nonspecific CD8 + T cells did not significantly contribute to protective immunity. In the livers of infected mice, specific CD8 + T cells expressed high levels of CD25, compatible with a local, activated effector phenotype. In vivo imaging of the liver revealed that specific CD8 + T cells interact with CD11c + cells around infected hepatocytes. The depletion of CD11c + cells virtually eliminated the clusters in the liver, leading to a significant decrease in protection. These experiments reveal an essential role of hepatic CD11c + dendritic cells and presumably macrophages in the formation of CD8 + T cell clusters around Plasmodium -infected hepatocytes. Once cluster formation is triggered by parasite-specific CD8 + T cells, specific and unrelated activated CD8 + T cells join the clusters in a chemokine- and dendritic cell-dependent manner. Nonspecific CD8 + T cells seem to play a limited role in protective immunity against Plasmodium parasites. Copyright © 2018 American Society for Microbiology.

  9. Nanoscale live cell optical imaging of the dynamics of intracellular microvesicles in neural cells.

    Science.gov (United States)

    Lee, Sohee; Heo, Chaejeong; Suh, Minah; Lee, Young Hee

    2013-11-01

    Recent advances in biotechnology and imaging technology have provided great opportunities to investigate cellular dynamics. Conventional imaging methods such as transmission electron microscopy, scanning electron microscopy, and atomic force microscopy are powerful techniques for cellular imaging, even at the nanoscale level. However, these techniques have limitations applications in live cell imaging because of the experimental preparation required, namely cell fixation, and the innately small field of view. In this study, we developed a nanoscale optical imaging (NOI) system that combines a conventional optical microscope with a high resolution dark-field condenser (Cytoviva, Inc.) and halogen illuminator. The NOI system's maximum resolution for live cell imaging is around 100 nm. We utilized NOI to investigate the dynamics of intracellular microvesicles of neural cells without immunocytological analysis. In particular, we studied direct, active random, and moderate random dynamic motions of intracellular microvesicles and visualized lysosomal vesicle changes after treatment of cells with a lysosomal inhibitor (NH4Cl). Our results indicate that the NOI system is a feasible, high-resolution optical imaging system for live small organelles that does not require complicated optics or immunocytological staining processes.

  10. Heparanase facilitates cell adhesion and spreading by clustering of cell surface heparan sulfate proteoglycans.

    Directory of Open Access Journals (Sweden)

    Flonia Levy-Adam

    2008-06-01

    Full Text Available Heparanase is a heparan sulfate (HS degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions. Non-enzymatic activities of heparanase include enhanced adhesion of tumor-derived cells and primary T-cells. Attempting to identify functional domains of heparanase that would serve as targets for drug development, we have identified heparin binding domains of heparanase. A corresponding peptide (residues Lys(158-Asp(171, termed KKDC was demonstrated to physically associate with heparin and HS, and to inhibit heparanase enzymatic activity. We hypothesized that the pro-adhesive properties of heparanase are mediated by its interaction with cell surface HS proteoglycans, and utilized the KKDC peptide to examine this possibility. We provide evidence that the KKDC peptide interacts with cell membrane HS, resulting in clustering of syndecan-1 and syndecan-4. We applied classical analysis of cell morphology, fluorescent and time-lapse microscopy and demonstrated that the KKDC peptide efficiently stimulates the adhesion and spreading of various cell types, mediated by PKC, Src, and the small GTPase Rac1. These results support, and further substantiate the notion that heparanase function is not limited to its enzymatic activity.

  11. Low-power, Confocal Imaging of Protein Localization in Living Cells (7214-150), Phase I

    Data.gov (United States)

    National Aeronautics and Space Administration — The proposed technology genetically labels intracellular structures and visualizes protein interactions in living cells using a compact, confocal microscope with...

  12. Method for Imaging Live-Cell RNA Using an RNA Aptamer and a Fluorescent Probe.

    Science.gov (United States)

    Sato, Shin-Ichi; Yatsuzuka, Kenji; Katsuda, Yousuke; Uesugi, Motonari

    2018-01-01

    Live-cell imaging of mRNA dynamics is increasingly important to understanding spatially restricted gene expression. We recently developed a convenient and versatile method that uses a gene-specific RNA aptamer and a fluorescent probe to enable spatiotemporal imaging of endogenous mRNAs in living cells. The method was validated by live-cell imaging of the endogenous mRNA of β-actin. The new RNA-imaging technology might be useful for live-cell imaging of any RNA molecules.

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

    Science.gov (United States)

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

    2016-01-01

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

  14. Harnessing molecular motors for nanoscale pulldown in live cells.

    Science.gov (United States)

    Bird, Jonathan E; Barzik, Melanie; Drummond, Meghan C; Sutton, Daniel C; Goodman, Spencer M; Morozko, Eva L; Cole, Stacey M; Boukhvalova, Alexandra K; Skidmore, Jennifer; Syam, Diana; Wilson, Elizabeth A; Fitzgerald, Tracy; Rehman, Atteeq U; Martin, Donna M; Boger, Erich T; Belyantseva, Inna A; Friedman, Thomas B

    2017-02-01

    Protein-protein interactions (PPIs) regulate assembly of macromolecular complexes, yet remain challenging to study within the native cytoplasm where they normally exert their biological effect. Here we miniaturize the concept of affinity pulldown, a gold-standard in vitro PPI interrogation technique, to perform nanoscale pulldowns (NanoSPDs) within living cells. NanoSPD hijacks the normal process of intracellular trafficking by myosin motors to forcibly pull fluorescently tagged protein complexes along filopodial actin filaments. Using dual-color total internal reflection fluorescence microscopy, we demonstrate complex formation by showing that bait and prey molecules are simultaneously trafficked and actively concentrated into a nanoscopic volume at the tips of filopodia. The resulting molecular traffic jams at filopodial tips amplify fluorescence intensities and allow PPIs to be interrogated using standard epifluorescence microscopy. A rigorous quantification framework and software tool are provided to statistically evaluate NanoSPD data sets. We demonstrate the capabilities of NanoSPD for a range of nuclear and cytoplasmic PPIs implicated in human deafness, in addition to dissecting these interactions using domain mapping and mutagenesis experiments. The NanoSPD methodology is extensible for use with other fluorescent molecules, in addition to proteins, and the platform can be easily scaled for high-throughput applications. © 2017 Bird, Barzik, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  15. Growing Bladder-Cancer Cells In Three-Dimensional Clusters

    Science.gov (United States)

    Spaulding, Glenn F.; Prewett, Tacey L.; Goodwin, Thomas J.

    1995-01-01

    Artificial growth process helps fill gaps in cancer research. Cell cultures more accurate as models for in vivo studies and as sources of seed cells for in vivo studies. Effected in horizontal rotating bioreactor described in companion article, "Simplified Bioreactor for Growing Mammalian Cells" (MSC-22060). Provides aggregates of cells needed to fill many of gaps.

  16. Nanometre-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

    2013-08-01

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

  17. Multi-scale Modeling of Chromosomal DNA in Living Cells

    Science.gov (United States)

    Spakowitz, Andrew

    The organization and dynamics of chromosomal DNA play a pivotal role in a range of biological processes, including gene regulation, homologous recombination, replication, and segregation. Establishing a quantitative theoretical model of DNA organization and dynamics would be valuable in bridging the gap between the molecular-level packaging of DNA and genome-scale chromosomal processes. Our research group utilizes analytical theory and computational modeling to establish a predictive theoretical model of chromosomal organization and dynamics. In this talk, I will discuss our efforts to develop multi-scale polymer models of chromosomal DNA that are both sufficiently detailed to address specific protein-DNA interactions while capturing experimentally relevant time and length scales. I will demonstrate how these modeling efforts are capable of quantitatively capturing aspects of behavior of chromosomal DNA in both prokaryotic and eukaryotic cells. This talk will illustrate that capturing dynamical behavior of chromosomal DNA at various length scales necessitates a range of theoretical treatments that accommodate the critical physical contributions that are relevant to in vivo behavior at these disparate length and time scales. National Science Foundation, Physics of Living Systems Program (PHY-1305516).

  18. 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.

  19. Cost-effectiveness of nurse-led multifactorial care to prevent or postpone new disabilities in community-living older people : Results of a cluster randomized trial

    NARCIS (Netherlands)

    Suijker, Jacqueline J.; MacNeil-Vroomen, Janet L.; van Rijn, Marjon; Buurman, Bianca M.; de Rooij, Sophia E.; van Charente, Eric P. Moll; Bosmans, Judith E.

    2017-01-01

    Objective To evaluate the cost-effectiveness of nurse-led multifactorial care to prevent or postpone new disabilities in community-living older people in comparison with usual care. Methods We conducted cost-effectiveness and cost-utility analyses alongside a cluster randomized trial with one-year

  20. Cost-effectiveness of nurse-led multifactorial care to prevent or postpone new disabilities in community-living older people: Results of a cluster randomized trial

    NARCIS (Netherlands)

    Suijker, Jacqueline J.; MacNeil-Vroomen, Janet L.; van Rijn, Marjon; Buurman, Bianca M.; de Rooij, Sophia E.; Moll van Charante, Eric P.; Bosmans, Judith E.

    2017-01-01

    Objective To evaluate the cost-effectiveness of nurse-led multifactorial care to prevent or postpone new disabilities in community-living older people in comparison with usual care. Methods We conducted cost-effectiveness and cost-utility analyses alongside a cluster randomized trial with one-year

  1. Cost-effectiveness of nurse-led multifactorial care to prevent or postpone new disabilities in community-living older people : Results of a cluster randomized trial

    NARCIS (Netherlands)

    Suijker, Jacqueline J; MacNeil-Vroomen, Janet L; van Rijn, Marjon; Buurman, Bianca M; de Rooij, Sophia E; Moll van Charante, Eric P; Bosmans, Judith E

    2017-01-01

    OBJECTIVE: To evaluate the cost-effectiveness of nurse-led multifactorial care to prevent or postpone new disabilities in community-living older people in comparison with usual care. METHODS: We conducted cost-effectiveness and cost-utility analyses alongside a cluster randomized trial with one-year

  2. An On-Line Oxygen Forecasting System for Waterless Live Transportation of Flatfish Based on Feature Clustering

    Directory of Open Access Journals (Sweden)

    Yongjun Zhang

    2017-09-01

    Full Text Available Accurate prediction of forthcoming oxygen concentration during waterless live fish transportation plays a key role in reducing the abnormal occurrence, increasing the survival rate in delivery operations, and optimizing manufacturing costs. The most effective ambient monitoring techniques that are based on the analysis of historical process data when performing forecasting operations do not fully consider current ambient influence. This is likely lead to a greater deviation in on-line oxygen level forecasting in real situations. Therefore, it is not advisable for the system to perform early warning and on-line air adjustment in delivery. In this paper, we propose a hybrid method and its implementation system that combines a gray model (GM (1, 1 with least squares support vector machines (LSSVM that can be used effectively as a forecasting model to perform early warning effectively according to the dynamic changes of oxygen in a closed system. For accurately forecasting of the oxygen level, the fuzzy C-means clustering (FCM algorithm was utilized for classification according to the flatfish’s physical features—i.e., length and weight—for more pertinent training. The performance of the gray model-particle swarm optimization-least squares support vector machines (GM-PSO-LSSVM model was compared with the traditional modeling approaches of GM (1, 1 and LSSVM by applying it to predict on-line oxygen level, and the results showed that its predictions were more accurate than those of the LSSVM and grey model. Therefore, it is a suitable and effective method for abnormal condition forecasting and timely control in the waterless live transportation of flatfish.

  3. DAFi: A directed recursive data filtering and clustering approach for improving and interpreting data clustering identification of cell populations from polychromatic flow cytometry data.

    Science.gov (United States)

    Lee, Alexandra J; Chang, Ivan; Burel, Julie G; Lindestam Arlehamn, Cecilia S; Mandava, Aishwarya; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro; Scheuermann, Richard H; Qian, Yu

    2018-04-17

    Computational methods for identification of cell populations from polychromatic flow cytometry data are changing the paradigm of cytometry bioinformatics. Data clustering is the most common computational approach to unsupervised identification of cell populations from multidimensional cytometry data. However, interpretation of the identified data clusters is labor-intensive. Certain types of user-defined cell populations are also difficult to identify by fully automated data clustering analysis. Both are roadblocks before a cytometry lab can adopt the data clustering approach for cell population identification in routine use. We found that combining recursive data filtering and clustering with constraints converted from the user manual gating strategy can effectively address these two issues. We named this new approach DAFi: Directed Automated Filtering and Identification of cell populations. Design of DAFi preserves the data-driven characteristics of unsupervised clustering for identifying novel cell subsets, but also makes the results interpretable to experimental scientists through mapping and merging the multidimensional data clusters into the user-defined two-dimensional gating hierarchy. The recursive data filtering process in DAFi helped identify small data clusters which are otherwise difficult to resolve by a single run of the data clustering method due to the statistical interference of the irrelevant major clusters. Our experiment results showed that the proportions of the cell populations identified by DAFi, while being consistent with those by expert centralized manual gating, have smaller technical variances across samples than those from individual manual gating analysis and the nonrecursive data clustering analysis. Compared with manual gating segregation, DAFi-identified cell populations avoided the abrupt cut-offs on the boundaries. DAFi has been implemented to be used with multiple data clustering methods including K-means, FLOCK, FlowSOM, and

  4. Differential migration and proliferation of geometrical ensembles of cell clusters

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Girish; Chen, Bo; Co, Carlos C.; Ho, Chia-Chi, E-mail: hocc@email.uc.edu

    2011-06-10

    Differential cell migration and growth drives the organization of specific tissue forms and plays a critical role in embryonic development, tissue morphogenesis, and tumor invasion. Localized gradients of soluble factors and extracellular matrix have been shown to modulate cell migration and proliferation. Here we show that in addition to these factors, initial tissue geometry can feedback to generate differential proliferation, cell polarity, and migration patterns. We apply layer by layer polyelectrolyte assembly to confine multicellular organization and subsequently release cells to demonstrate the spatial patterns of cell migration and growth. The cell shapes, spreading areas, and cell-cell contacts are influenced strongly by the confining geometry. Cells within geometric ensembles are morphologically polarized. Symmetry breaking was observed for cells on the circular pattern and cells migrate toward the corners and in the direction parallel to the longest dimension of the geometric shapes. This migration pattern is disrupted when actomyosin based tension was inhibited. Cells near the edge or corner of geometric shapes proliferate while cells within do not. Regions of higher rate of cell migration corresponded to regions of concentrated growth. These findings demonstrate that multicellular organization can result in spatial patterns of migration and proliferation.

  5. Differential migration and proliferation of geometrical ensembles of cell clusters

    International Nuclear Information System (INIS)

    Kumar, Girish; Chen, Bo; Co, Carlos C.; Ho, Chia-Chi

    2011-01-01

    Differential cell migration and growth drives the organization of specific tissue forms and plays a critical role in embryonic development, tissue morphogenesis, and tumor invasion. Localized gradients of soluble factors and extracellular matrix have been shown to modulate cell migration and proliferation. Here we show that in addition to these factors, initial tissue geometry can feedback to generate differential proliferation, cell polarity, and migration patterns. We apply layer by layer polyelectrolyte assembly to confine multicellular organization and subsequently release cells to demonstrate the spatial patterns of cell migration and growth. The cell shapes, spreading areas, and cell-cell contacts are influenced strongly by the confining geometry. Cells within geometric ensembles are morphologically polarized. Symmetry breaking was observed for cells on the circular pattern and cells migrate toward the corners and in the direction parallel to the longest dimension of the geometric shapes. This migration pattern is disrupted when actomyosin based tension was inhibited. Cells near the edge or corner of geometric shapes proliferate while cells within do not. Regions of higher rate of cell migration corresponded to regions of concentrated growth. These findings demonstrate that multicellular organization can result in spatial patterns of migration and proliferation.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2011-01-01

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

  8. Freeform micropatterning of living cells into cell culture medium using direct inkjet printing.

    Science.gov (United States)

    Park, Ju An; Yoon, Sejeong; Kwon, Jimin; Now, Hesung; Kim, Young Kwon; Kim, Woo-Jong; Yoo, Joo-Yeon; Jung, Sungjune

    2017-11-06

    Microfabrication methods have widely been used to control the local cellular environment on a micron scale. However, accurately mimicking the complexity of the in vivo tissue architecture while maintaining the freedom of form and design is still a challenge when co-culturing multiple types of cells on the same substrate. For the first time, we present a drop-on-demand inkjet printing method to directly pattern living cells into a cell-friendly liquid environment. High-resolution control of cell location is achieved by precisely optimizing printing parameters with high-speed imaging of cell jetting and impacting behaviors. We demonstrated the capabilities of the direct cell printing method by co-printing different cells into various designs, including complex gradient arrangements. Finally, we applied this technique to investigate the influence of the heterogeneity and geometry of the cell population on the infectivity of seasonal H1N1 influenza virus (PR8) by generating A549 and HeLa cells printed in checkboard patterns of different sizes in a medium-filled culture dish. Direct inkjet cell patterning can be a powerful and versatile tool for both fundamental biology and applied biotechnology.

  9. 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.

  10. Clustered localization of STAT3 during the cell cycle detected by super-resolution fluorescence microscopy

    Science.gov (United States)

    Gao, Jing; Chen, Junling; Cai, Mingjun; Xu, Haijiao; Jiang, Junguang; Tong, Ti; Wang, Hongda

    2017-06-01

    Signal transducer and activator of transcription 3 (STAT3) plays a key role in various cellular processes such as cell proliferation, differentiation, apoptosis and immune responses. In particular, STAT3 has emerged as a potential molecular target for cancer therapy. The functional role and standard activation mechanism of STAT3 have been well studied, however, the spatial distribution of STAT3 during the cell cycle is poorly known. Therefore, it is indispensable to study STAT3 spatial arrangement and nuclear-cytoplasimic localization at the different phase of cell cycle in cancer cells. By direct stochastic optical reconstruction microscopy imaging, we find that STAT3 forms various number and size of clusters at the different cell-cycle stage, which could not be clearly observed by conventional fluorescent microscopy. STAT3 clusters get more and larger gradually from G1 to G2 phase, during which time transcription and other related activities goes on consistently. The results suggest that there is an intimate relationship between the clustered characteristic of STAT3 and the cell-cycle behavior. Meanwhile, clustering would facilitate STAT3 rapid response to activating signals due to short distances between molecules. Our data might open a new door to develop an antitumor drug for inhibiting STAT3 signaling pathway by destroying its clusters.

  11. Half-lives for proton emission, alpha decay, cluster radioactivity, and cold fission processes calculated in a unified theoretical framework

    Energy Technology Data Exchange (ETDEWEB)

    Duarte, S.B.; Tavares, O.A.P.; Guzman, F.; Dimarco, A. [Centro Brasileiro de Pesquisas Fisicas (CBPF), Rio de Janeiro, RJ (Brazil); Garcia, F. [Sao Paulo Univ., SP (Brazil). Inst. de Fisica; Universidade Estadual de Santa Cruz, Ilheus, BA (Brazil). Dept. de Ciencias Exatas e Tecnologicas; Rodriguez, O. [Sao Paulo Univ., SP (Brazil). Inst. de Fisica; Instituto Superior de Ciencias e Tecnologia Nucleares, La Habana (Cuba); Goncalves, M. [Instituto de Radioprotecao e Dosimetria (IRD), Rio de Janeiro, RJ (Brazil)

    2002-01-01

    Half-life values of spontaneous nuclear decay processes are presented in the framework of the Effective Liquid Drop Model (ELDM) using the combination of varying mass asymmetry shape description for the mass transfer with Werner-Wheeler's inertia coefficient V{sub MAS}/WW. The calculated half-lives of ground-state to ground-state transitions for the proton emission, alpha decay, cluster radioactivity, and cold fission processes are compared with experimental data. Results have shown that the ELDM is a very efficient model to describe these different decay processes in a same, unified theoretical framework. A Table listing the predicted half-life values, {tau}{sub c} is presented for all possible cases of spontaneous nuclear break-up such that -7.30 <{approx_equal} log{sub 10} {tau}{sub c} [S] <{approx_equal} 27.50 and log {sub 10}({tau}/{tau}{sub c}) > -17.0, where {tau} is the total half-life of the parent nucleus. (author)

  12. Phenotype Clustering of Breast Epithelial Cells in Confocal Imagesbased on Nuclear Protein Distribution Analysis

    Energy Technology Data Exchange (ETDEWEB)

    Long, Fuhui; Peng, Hanchuan; Sudar, Damir; Levievre, Sophie A.; Knowles, David W.

    2006-09-05

    Background: The distribution of the chromatin-associatedproteins plays a key role in directing nuclear function. Previously, wedeveloped an image-based method to quantify the nuclear distributions ofproteins and showed that these distributions depended on the phenotype ofhuman mammary epithelial cells. Here we describe a method that creates ahierarchical tree of the given cell phenotypes and calculates thestatistical significance between them, based on the clustering analysisof nuclear protein distributions. Results: Nuclear distributions ofnuclear mitotic apparatus protein were previously obtained fornon-neoplastic S1 and malignant T4-2 human mammary epithelial cellscultured for up to 12 days. Cell phenotype was defined as S1 or T4-2 andthe number of days in cultured. A probabilistic ensemble approach wasused to define a set of consensus clusters from the results of multipletraditional cluster analysis techniques applied to the nucleardistribution data. Cluster histograms were constructed to show how cellsin any one phenotype were distributed across the consensus clusters.Grouping various phenotypes allowed us to build phenotype trees andcalculate the statistical difference between each group. The resultsshowed that non-neoplastic S1 cells could be distinguished from malignantT4-2 cells with 94.19 percent accuracy; that proliferating S1 cells couldbe distinguished from differentiated S1 cells with 92.86 percentaccuracy; and showed no significant difference between the variousphenotypes of T4-2 cells corresponding to increasing tumor sizes.Conclusion: This work presents a cluster analysis method that canidentify significant cell phenotypes, based on the nuclear distributionof specific proteins, with high accuracy.

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

    Science.gov (United States)

    Pouyan, Maziyar Baran; Nourani, Mehrdad

    2017-07-01

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

  14. Application Of WIMS Code To Calculation Kartini Reactor Parameters By Pin-Cell And Cluster Method

    International Nuclear Information System (INIS)

    Sumarsono, Bambang; Tjiptono, T.W.

    1996-01-01

    Analysis UZrH fuel element parameters calculation in Kartini Reactor by WIMS Code has been done. The analysis is done by pin cell and cluster method. The pin cell method is done as a function percent burn-up and by 8 group 3 region analysis and cluster method by 8 group 12 region analysis. From analysis and calculation resulted K ∼ = 1.3687 by pin cell method and K ∼ = 1.3162 by cluster method and so deviation is 3.83%. By pin cell analysis as a function percent burn-up at the percent burn-up greater than 59.50%, the multiplication factor is less than one (k ∼ < 1) it is mean that the fuel element reactivity is negative

  15. In vivo UVB irradiation induces clustering of Fas (CD95) on human epidermal cells

    DEFF Research Database (Denmark)

    Bang, Bo; Gniadecki, Robert; Larsen, Jørgen K

    2003-01-01

    In vitro studies with human cell lines have demonstrated that the death receptor Fas plays a role in ultraviolet (UV)-induced apoptosis. The purpose of the present study was to investigate the relation between Fas expression and apoptosis as well as clustering of Fas in human epidermis after...... clustering has a functional significance. Our results ar in accordance with previous findings from in vitro studies, and suggest that Fas is activated in vivo in human epidermis after UVB exposure....

  16. Clarification of mural cell coverage of vascular endothelial cells by live imaging of zebrafish.

    Science.gov (United States)

    Ando, Koji; Fukuhara, Shigetomo; Izumi, Nanae; Nakajima, Hiroyuki; Fukui, Hajime; Kelsh, Robert N; Mochizuki, Naoki

    2016-04-15

    Mural cells (MCs) consisting of vascular smooth muscle cells and pericytes cover the endothelial cells (ECs) to regulate vascular stability and homeostasis. Here, we clarified the mechanism by which MCs develop and cover ECs by generating transgenic zebrafish lines that allow live imaging of MCs and by lineage tracing in vivo To cover cranial vessels, MCs derived from either neural crest cells or mesoderm emerged around the preformed EC tubes, proliferated and migrated along EC tubes. During their migration, the MCs moved forward by extending their processes along the inter-EC junctions, suggesting a role for inter-EC junctions as a scaffold for MC migration. In the trunk vasculature, MCs derived from mesoderm covered the ventral side of the dorsal aorta (DA), but not the posterior cardinal vein. Furthermore, the MCs migrating from the DA or emerging around intersegmental vessels (ISVs) preferentially covered arterial ISVs rather than venous ISVs, indicating that MCs mostly cover arteries during vascular development. Thus, live imaging and lineage tracing enabled us to clarify precisely how MCs cover the EC tubes and to identify the origins of MCs. © 2016. Published by The Company of Biologists Ltd.

  17. 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

  18. Perbandingan Quantum Clustering Dan Support Vector Clustering Untuk Data Microarray Expression Yeast Cell Dalam Ruang Singular Value Decomposition (Svd)

    OpenAIRE

    Riwinoto

    2012-01-01

    Sekarang ini, metode clustering telah diimplementasikan dalam riset DNA. Data dari DNA didapat melalui teknik microarray. Dengan menggunakan metode teknik SVD, dimensi data dikurangi sehingga mempermudah proses komputasi. Dalam paper ini, ditampilkan hasil clustering tanpa pengarahan terhadap gen-gen dari data bakteri ragi dengan menggunakan metode quantum clustering. Sebagai pembanding, dilakukan juga clustering menggunakan metoda Support Vector Clustering. Selain itu juga ditampilkan data h...

  19. 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.

  20. A nonemissive iridium(III) complex that specifically lights-up the nuclei of living cells.

    Science.gov (United States)

    Li, Chunyan; Yu, Mengxiao; Sun, Yun; Wu, Yongquan; Huang, Chunhui; Li, Fuyou

    2011-07-27

    A nonemissive cyclometalated iridium(III) solvent complex, without conjugation with a cell-penetrating molecular transporter, [Ir(ppy)(2)(DMSO)(2)](+)PF(6)(-) (LIr1), has been developed as a first reaction-based fluorescence-turn-on agent for the nuclei of living cells. LIr1 can rapidly and selectively light-up the nuclei of living cells over fixed cells, giving rise to a significant luminescence enhancement (200-fold), and shows very low cytotoxicity at the imaging concentration (incubation time atomic emission spectroscopy (ICP-AEC), LIr1 is accumulated in the nuclei of living cells and converted into an intensely emissive adduct. Such novel reaction-based nuclear staining for visualizing exclusively the nuclei of living cells with a significant luminescence enhancement may extend the arsenal of currently available fluorescent stains for specific staining of cellular compartments.

  1. On the coherent behavior of pancreatic beta cell clusters

    International Nuclear Information System (INIS)

    Loppini, Alessandro; Capolupo, Antonio; Cherubini, Christian; Gizzi, Alessio; Bertolaso, Marta; Filippi, Simonetta; Vitiello, Giuseppe

    2014-01-01

    Beta cells in pancreas represent an example of coupled biological oscillators which via communication pathways, are able to synchronize their electrical activity, giving rise to pulsatile insulin release. In this work we numerically analyze scale free self-similarity features of membrane voltage signal power density spectrum, through a stochastic dynamical model for beta cells in the islets of Langerhans fine tuned on mouse experimental data. Adopting the algebraic approach of coherent state formalism, we show how coherent molecular domains can arise from proper functional conditions leading to a parallelism with “phase transition” phenomena of field theory. - Highlights: • Beta cells in pancreas are coupled oscillators able to synchronize their activity. • We analyze scale free self-similarity features for beta cells. • We adopt the algebraic approach of coherent state formalism. • We show that coherent molecular domains arise from functional conditions

  2. A Morphological identification cell cytotoxicity assay using cytoplasm-localized fluorescent probe (CLFP) to distinguish living and dead cells.

    Science.gov (United States)

    Lai, Fangfang; Shen, Zhengwei; Wen, Hui; Chen, Jialing; Zhang, Xiang; Lin, Ping; Yin, Dali; Cui, Huaqing; Chen, Xiaoguang

    2017-01-08

    Cell cytotoxicity assays include cell activity assays and morphological identification assays. Currently, all frequently used cytotoxicity assays belong to cell activity assays but suffer from detection limitations. Morphological identification of cell death remains as the gold standard, although the method is difficult to scale up. At present there is no generally accepted morphological identification based cell cytotoxicity assay. In this study, we applied previous developed cell cytoplasm-localized fluorescent probe (CLFP) to display cell morphologies. Under fluorescence microscopy, the fluorescence morphology and intensity of living cells are distinct from dead cells. Based on these characters we extracted the images of living cells from series of samples via computational analysis. Thus, a novel cell morphological identification cytotoxicity assay (CLFP assay) is developed. The performance of the CLFP assay was similar to cell activity assay (MTT assay), but the accuracy of the CLFP assay was superior when measuring the cytotoxicity of active compounds. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Perspective on Circulating Tumor Cell Clusters: Why It Takes a Village to Metastasize.

    Science.gov (United States)

    Giuliano, Mario; Shaikh, Anum; Lo, Hin Ching; Arpino, Grazia; De Placido, Sabino; Zhang, Xiang H; Cristofanilli, Massimo; Schiff, Rachel; Trivedi, Meghana V

    2018-02-15

    Circulating tumor cell (CTC) clusters may represent one of the key mechanisms initiating the metastasis process. However, the series of pathophysiologic events by which CTC clusters originate, enter the circulation, and reach the distant sites remain to be identified. The cellular and molecular mechanisms that provide survival advantage for CTC clusters during the transit in the blood stream are also still largely unknown. Understanding the biology of CTC clusters is critical to assess this unified scheme employed by cancer and to device strategies to overcome key pathways responsible for their improved metastatic potential. CTC clusters remain an underdeveloped area of research begging the attention of multidisciplinary cancer research teams. Here, we provide insight on existing preclinical evidence on the potential mechanisms leading to CTC cluster formation and dissemination and on processes that may offer survival advantage. We also offer our perspective on future directions to delineate the role of CTC clusters in metastatic cascade and discuss their clinical significance. Cancer Res; 78(4); 845-52. ©2018 AACR . ©2018 American Association for Cancer Research.

  5. A single HIV-1 cluster and a skewed immune homeostasis drive the early spread of HIV among resting CD4+ cell subsets within one month post-infection.

    Directory of Open Access Journals (Sweden)

    Charline Bacchus

    Full Text Available Optimizing therapeutic strategies for an HIV cure requires better understanding the characteristics of early HIV-1 spread among resting CD4+ cells within the first month of primary HIV-1 infection (PHI. We studied the immune distribution, diversity, and inducibility of total HIV-DNA among the following cell subsets: monocytes, peripheral blood activated and resting CD4 T cells, long-lived (naive [TN] and central-memory [TCM] and short-lived (transitional-memory [TTM] and effector-memory cells [TEM] resting CD4+T cells from 12 acutely-infected individuals recruited at a median 36 days from infection. Cells were sorted for total HIV-DNA quantification, phylogenetic analysis and inducibility, all studied in relation to activation status and cell signaling. One month post-infection, a single CCR5-restricted viral cluster was massively distributed in all resting CD4+ subsets from 88% subjects, while one subject showed a slight diversity. High levels of total HIV-DNA were measured among TN (median 3.4 log copies/million cells, although 10-fold less (p = 0.0005 than in equally infected TCM (4.5, TTM (4.7 and TEM (4.6 cells. CD3-CD4+ monocytes harbored a low viral burden (median 2.3 log copies/million cells, unlike equally infected resting and activated CD4+ T cells (4.5 log copies/million cells. The skewed repartition of resting CD4 subsets influenced their contribution to the pool of resting infected CD4+T cells, two thirds of which consisted of short-lived TTM and TEM subsets, whereas long-lived TN and TCM subsets contributed the balance. Each resting CD4 subset produced HIV in vitro after stimulation with anti-CD3/anti-CD28+IL-2 with kinetics and magnitude varying according to subset differentiation, while IL-7 preferentially induced virus production from long-lived resting TN cells. In conclusion, within a month of infection, a clonal HIV-1 cluster is massively distributed among resting CD4 T-cell subsets with a flexible inducibility

  6. A Single HIV-1 Cluster and a Skewed Immune Homeostasis Drive the Early Spread of HIV among Resting CD4+ Cell Subsets within One Month Post-Infection

    Science.gov (United States)

    Avettand-Fenoël, Véronique; Nembot, Georges; Mélard, Adeline; Blanc, Catherine; Lascoux-Combe, Caroline; Slama, Laurence; Allegre, Thierry; Allavena, Clotilde; Yazdanpanah, Yazdan; Duvivier, Claudine; Katlama, Christine; Goujard, Cécile; Seksik, Bao Chau Phung; Leplatois, Anne; Molina, Jean-Michel; Meyer, Laurence; Autran, Brigitte; Rouzioux, Christine

    2013-01-01

    Optimizing therapeutic strategies for an HIV cure requires better understanding the characteristics of early HIV-1 spread among resting CD4+ cells within the first month of primary HIV-1 infection (PHI). We studied the immune distribution, diversity, and inducibility of total HIV-DNA among the following cell subsets: monocytes, peripheral blood activated and resting CD4 T cells, long-lived (naive [TN] and central-memory [TCM]) and short-lived (transitional-memory [TTM] and effector-memory cells [TEM]) resting CD4+T cells from 12 acutely-infected individuals recruited at a median 36 days from infection. Cells were sorted for total HIV-DNA quantification, phylogenetic analysis and inducibility, all studied in relation to activation status and cell signaling. One month post-infection, a single CCR5-restricted viral cluster was massively distributed in all resting CD4+ subsets from 88% subjects, while one subject showed a slight diversity. High levels of total HIV-DNA were measured among TN (median 3.4 log copies/million cells), although 10-fold less (p = 0.0005) than in equally infected TCM (4.5), TTM (4.7) and TEM (4.6) cells. CD3−CD4+ monocytes harbored a low viral burden (median 2.3 log copies/million cells), unlike equally infected resting and activated CD4+ T cells (4.5 log copies/million cells). The skewed repartition of resting CD4 subsets influenced their contribution to the pool of resting infected CD4+T cells, two thirds of which consisted of short-lived TTM and TEM subsets, whereas long-lived TN and TCM subsets contributed the balance. Each resting CD4 subset produced HIV in vitro after stimulation with anti-CD3/anti-CD28+IL-2 with kinetics and magnitude varying according to subset differentiation, while IL-7 preferentially induced virus production from long-lived resting TN cells. In conclusion, within a month of infection, a clonal HIV-1 cluster is massively distributed among resting CD4 T-cell subsets with a flexible inducibility, suggesting that

  7. 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.

  8. Dynamics of dye release from nanocarriers of different types in model cell membranes and living cells

    Directory of Open Access Journals (Sweden)

    Tkacheva T. N.

    2014-07-01

    Full Text Available Aim. To study the dynamics of lipophilic content release from nanocarriers of different types, organic molecular ensembles and inorganic nanoparticles (NPs in vitro experiments. Methods. Two-channel ratiometric fluorescence detection method based on Forster Resonance Energy Transfer, fluorescent spectroscopy and micro-spectroscopy have been used. Results. It has been found that the profiles of lipophilic dyes release from organic nanocarriers (PC liposomes and SDS micelles and inorganic ones (GdYVO4:Eu3+ and CeO2 NPs are well fitted by the first-order reaction kinetics in both model cell membranes and living cells (rat hepatocytes. The dye release constants (K and half-lives (t1/2 were analyzed. Conclusions. GdYVO4:Eu3+ and CeO2 NPs have been shown to provide faster lipophilic content release in model cell membranes as compared to PC liposomes. Negatively charged or lipophilic compounds added into nanocarriers can decrease the rate of lipophilic dyes release. Specific interaction of GdYVO4:Eu3+ NPs with rat hepatocytes has been observed.

  9. Automated detection and tracking of many cells by using 4D live-cell imaging data.

    Science.gov (United States)

    Tokunaga, Terumasa; Hirose, Osamu; Kawaguchi, Shotaro; Toyoshima, Yu; Teramoto, Takayuki; Ikebata, Hisaki; Kuge, Sayuri; Ishihara, Takeshi; Iino, Yuichi; Yoshida, Ryo

    2014-06-15

    Automated fluorescence microscopes produce massive amounts of images observing cells, often in four dimensions of space and time. This study addresses two tasks of time-lapse imaging analyses; detection and tracking of the many imaged cells, and it is especially intended for 4D live-cell imaging of neuronal nuclei of Caenorhabditis elegans. The cells of interest appear as slightly deformed ellipsoidal forms. They are densely distributed, and move rapidly in a series of 3D images. Thus, existing tracking methods often fail because more than one tracker will follow the same target or a tracker transits from one to other of different targets during rapid moves. The present method begins by performing the kernel density estimation in order to convert each 3D image into a smooth, continuous function. The cell bodies in the image are assumed to lie in the regions near the multiple local maxima of the density function. The tasks of detecting and tracking the cells are then addressed with two hill-climbing algorithms. The positions of the trackers are initialized by applying the cell-detection method to an image in the first frame. The tracking method keeps attacking them to near the local maxima in each subsequent image. To prevent the tracker from following multiple cells, we use a Markov random field (MRF) to model the spatial and temporal covariation of the cells and to maximize the image forces and the MRF-induced constraint on the trackers. The tracking procedure is demonstrated with dynamic 3D images that each contain >100 neurons of C.elegans. http://daweb.ism.ac.jp/yoshidalab/crest/ismb2014 SUPPLEMENTARY INFORMATION: Supplementary data are available at http://daweb.ism.ac.jp/yoshidalab/crest/ismb2014 © The Author 2014. Published by Oxford University Press.

  10. 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.

  11. 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

  12. 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.

  13. Using Cell Culture Models of Centrosome Amplification to Study Centrosome Clustering in Cancer.

    Science.gov (United States)

    Kwon, Mijung

    2016-01-01

    The link between centrosome amplification and cancer has been recognized for more than a century, raising many key questions about the biology of both normal and cancer cells. In particular, the presence of extra centrosomes imposes a great challenge to a dividing cell by increasing the likelihood of catastrophic multipolar divisions. Only recently have we begun to understand how cancer cells successfully divide by clustering their extra centrosomes for bipolar division. Several hurdles to dissecting centrosome clustering include limitations in the methodologies used to quantify centrosome amplification, and the lack of appropriate cell culture models. Here, we describe how to accurately assess centrosome number and create isogenic cell lines with or without centrosome amplification. We then describe how imaging of cell division in these cell culture models leads to identification of the molecular machinery uniquely required for cells with extra centrosomes. These approaches have led to the identification of molecular targets for selective cancer therapeutics that can kill cancer cells with extra centrosomes without affecting normal cells with two centrosomes. We further anticipate that the approaches described here will be broadly applicable for studying the causes and consequences of centrosome amplification in a variety of contexts across cancer pathophysiology, such as cell migration and metastasis.

  14. The first successful observation of in-cell NMR signals of DNA and RNA in living human cells.

    Science.gov (United States)

    Yamaoki, Yudai; Kiyoishi, Ayaka; Miyake, Masayuki; Kano, Fumi; Murata, Masayuki; Nagata, Takashi; Katahira, Masato

    2018-01-31

    In order to understand intracellular biological events, information on the structure, dynamics and interaction of proteins and nucleic acids in living cells is of crucial importance. In-cell NMR is a promising method to obtain this information. Although NMR signals of proteins in human cells have been reported, those of nucleic acids were reported only in Xenopus laevis oocytes, i.e., not in human cells. Here, DNA and RNA were introduced into human cells by means of pore formation by bacterial toxin streptolysin O and subsequent resealing. Then, NMR signals of DNA and RNA were successfully observed for the first time in living human cells. The observed signals directly suggested the formation of DNA and RNA hairpin structures in living human cells.

  15. 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.

  16. Differences and homologies of chromosomal alterations within and between breast cancer cell lines: a clustering analysis.

    Science.gov (United States)

    Rondón-Lagos, Milena; Verdun Di Cantogno, Ludovica; Marchiò, Caterina; Rangel, Nelson; Payan-Gomez, Cesar; Gugliotta, Patrizia; Botta, Cristina; Bussolati, Gianni; Ramírez-Clavijo, Sandra R; Pasini, Barbara; Sapino, Anna

    2014-01-23

    The MCF7 (ER+/HER2-), T47D (ER+/HER2-), BT474 (ER+/HER2+) and SKBR3 (ER-/HER2+) breast cancer cell lines are widely used in breast cancer research as paradigms of the luminal and HER2 phenotypes. Although they have been subjected to cytogenetic analysis, their chromosomal abnormalities have not been carefully characterized, and their differential cytogenetic profiles have not yet been established. In addition, techniques such as comparative genomic hybridization (CGH), microarray-based CGH and multiplex ligation-dependent probe amplification (MLPA) have described specific regions of gains, losses and amplifications of these cell lines; however, these techniques cannot detect balanced chromosomal rearrangements (e.g., translocations or inversions) or low frequency mosaicism. A range of 19 to 26 metaphases of the MCF7, T47D, BT474 and SKBR3 cell lines was studied using conventional (G-banding) and molecular cytogenetic techniques (multi-color fluorescence in situ hybridization, M-FISH). We detected previously unreported chromosomal changes and determined the content and frequency of chromosomal markers. MCF7 and T47D (ER+/HER2-) cells showed a less complex chromosomal make up, with more numerical than structural alterations, compared to BT474 and SKBR3 (HER2+) cells, which harbored the highest frequency of numerical and structural aberrations. Karyotype heterogeneity and clonality were determined by comparing all metaphases within and between the four cell lines by hierarchical clustering. The latter analysis identified five main clusters. One of these clusters was characterized by numerical chromosomal abnormalities common to all cell lines, and the other four clusters encompassed cell-specific chromosomal abnormalities. T47D and BT474 cells shared the most chromosomal abnormalities, some of which were shared with SKBR3 cells. MCF7 cells showed a chromosomal pattern that was markedly different from those of the other cell lines. Our study provides a comprehensive

  17. 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.

  18. 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

  19. 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

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

    Science.gov (United States)

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

    2013-01-01

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

  1. Phosphorescent imaging of living cells using a cyclometalated iridium (III complex.

    Directory of Open Access Journals (Sweden)

    Dik-Lung Ma

    Full Text Available A cell permeable cyclometalated iridium(III complex has been developed as a phosphorescent probe for cell imaging. The iridium(III solvato complex [Ir(phq2(H2O]2] preferentially stains the cytoplasm of both live and dead cells with a bright luminescence.

  2. Digital autoradiography of human living cells with a MWPC

    International Nuclear Information System (INIS)

    Bellazzini, R.; Del Guerra, A.; Massai, M.M.; Ragadini, M.; Spandre, G.; Tonelli, G.

    1982-01-01

    The possibility of using a MWPC for the non-destructive identification of DNA repair deficient mammalian cells is discussed. The detection system relies on mapping the differential incorporation of a radioactive precursor of DNA biosynthesis by aggregates of cells. The resolving power, sensitivity and linearity of developed system are shown together with preliminary analogically and digitally reconstructed maps of 14 C or 3 H labelled cells

  3. Functional and immunohistochemical evaluation of porcine neonatal islet-like cell clusters

    DEFF Research Database (Denmark)

    Nielsen, T B; Yderstraede, K B; Schrøder, H D

    2003-01-01

    Porcine neonatal islet-like cell clusters (NICCs) may be an attractive source of insulin-producing tissue for xenotransplantation in type I diabetic patients. We examined the functional and immunohistochemical outcome of the islet grafts in vitro during long-term culture and in vivo after transpl...

  4. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

    Directory of Open Access Journals (Sweden)

    Monica Salamone

    Full Text Available In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4 and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs or Serine Integral Membrane Peptidases (SIMPs caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

  5. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

    Science.gov (United States)

    Salamone, Monica; Carfì Pavia, Francesco; Ghersi, Giulio

    2016-01-01

    In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

  6. Randomized evaluation of live attenuated vs. inactivated influenza vaccines in schools (RELATIVES) pilot study: a cluster randomized trial.

    Science.gov (United States)

    Kwong, Jeffrey C; Pereira, Jennifer A; Quach, Susan; Pellizzari, Rosana; Dusome, Edwina; Russell, Margaret L; Hamid, Jemila S; Feinberg, Yael; Winter, Anne-Luise; Gubbay, Jonathan B; Sirtonski, Brittany; Moher, Deanna; Sider, Doug; Finkelstein, Michael; Loeb, Mark

    2015-01-15

    School-based influenza immunization can effectively address accessibility barriers, but injected inactivated influenza vaccines (IIV) may not be acceptable to some children and parents in school settings. To better understand the feasibility of offering intranasal live attenuated influenza vaccines (LAIV) through schools, we assessed uptake, stakeholder acceptability, and cost of school-based delivery of LAIV compared to IIV. We piloted an open-label cluster randomized trial involving 10 elementary schools in Peterborough, Ontario during the 2013-2014 influenza vaccination campaign. Schools were randomized to having students receive IIV or LAIV at publicly-funded school-based clinics organized by the local public health department. We measured the percentage of students vaccinated with at least one dose of influenza vaccine at school. Stakeholder acceptability was evaluated through a questionnaire of parents and interviews of public health department personnel and school principals. We compared the costs per dose of vaccine administered, including staff time and costs of vaccines and supplies. Single-dose influenza vaccine uptake was higher for the five schools offering LAIV than for the five offering IIV (19.3% vs. 12.2%, p=0.02). Interviews with nine school principals and five public health department personnel suggested that the clinics ran smoothly with little disruption to school routines, and that LAIV was associated with increased efficiency and calmer children. All interviewees cited unfamiliarity with LAIV and the study recruitment package length as potential reasons for low uptake. The cost per vaccine dose administered was $38.67 for IIV and $43.50 for LAIV. Use of LAIV in school-based clinics was associated with increased vaccine uptake and the perception among immunizing staff of reduced child anxiety, but also slightly higher vaccine administration costs, compared to IIV. However, uptake was low for both groups. More effective strategies to promote

  7. Serving and Rendering Cluster-Based Ocean Model Output on a Geowall Using the Live Access Server

    Science.gov (United States)

    Moore, C. W.; Hermann, A. J.; Dobbins, E. L.

    2004-12-01

    Scientists at NOAA's Pacific Marine Environmental Laboratory are relying more and more on supercomputing platforms for their modeling efforts. Running ocean models on these large cluster machines poses problems in that domain sizes are increasing and tracking how the model dynamics are developing during a run requires high-bandwidth network time. In an effort to streamline this procedure both server and 3-D rending technology are utilized. Intermediate model results saved in netCDF file format can be served remotely to query model progress using the Live Access Server (LAS). In our implementation, a crontab script checks for model results and generates an XML data-file descriptor and adds the data set to the list of those available for LAS to serve up. On top of the default product choices (2-D plots, data listings, etc), the user can also chose one of two 3D file formats: either a VRML or a Vis5D file of the variable of interest. The LAS is built upon the Ferret data analysis package with the ability to re-grid variables defined on curvilinear coordinate grids and to serve up Vis5D files. An alternate back-end, written using the open-source Visualization Toolkit (VTK), can serve a VRML isosurface as well as current vector fields, keeping bandwidth low by utilizing topology-preserving polygon mesh decimation algorithms. Files served through our LAS system can be projected in passive stereo using a Geowall (www.geowall.org) by either Vis5D, or by ImmersaView. While ImmersaView offers the ability to animate through the VRML isosurfaces in collaboration with a remote researcher, Vis5D (an older-technology application) gives the user the ability to explore the data more thoroughly by allowing the scientist to change isosurfaces levels, or to probe the data using contour or vector slices. We will explore the possibility of using LAS as the server for the parallel, composite-rendering application ParaView.

  8. Engineered core-shell magnetic nanoparticle for MR dual-modal tracking and safe magnetic manipulation of ependymal cells in live rodents

    Science.gov (United States)

    Peng, Yung-Kang; Lui, Cathy N. P.; Chen, Yu-Wei; Chou, Shang-Wei; Chou, Pi-Tai; Yung, Ken K. L.; Edman Tsang, S. C.

    2018-01-01

    Tagging recognition group(s) on superparamagnetic iron oxide is known to aid localisation (imaging), stimulation and separation of biological entities using magnetic resonance imaging (MRI) and magnetic agitation/separation (MAS) techniques. Despite the wide applicability of iron oxide nanoparticles in T 2-weighted MRI and MAS, the quality of the images and safe manipulation of the exceptionally delicate neural cells in a live brain are currently the key challenges. Here, we demonstrate the engineered manganese oxide clusters-iron oxide core-shell nanoparticle as an MR dual-modal contrast agent for neural stem cells (NSCs) imaging and magnetic manipulation in live rodents. As a result, using this engineered nanoparticle and associated technologies, identification, stimulation and transportation of labelled potentially multipotent NSCs from a specific location of a live brain to another by magnetic means for self-healing therapy can therefore be made possible.

  9. Live-attenuated measles virus vaccine targets dendritic cells and macrophages in muscle of nonhuman primates

    NARCIS (Netherlands)

    L.J. Rennick (Linda); R.D. de Vries (Rory); T.J. Carsillo (Thomas J.); K. Lemon (Ken); G. van Amerongen (Geert); M. Ludlow (Martin); D.T. Nguyen (Tien); S. Yüksel (Selma); R.J. Verbugh (Joyce); P. Haddock (Paula); S. McQuaid (Stephen); W.P. Duprex (Paul); R.L. de Swart (Rik)

    2015-01-01

    textabstractAlthough live-attenuated measles virus (MV) vaccines have been used successfully for over 50 years, the target cells that sustain virus replication in vivo are still unknown. We generated a reverse genetics system for the live-attenuated MV vaccine strain Edmonston- Zagreb (EZ), allowing

  10. Human omentum fat-derived mesenchymal stem cells transdifferentiates into pancreatic islet-like cluster.

    Science.gov (United States)

    Dhanasekaran, M; Indumathi, S; Harikrishnan, R; Mishra, Rashmi; Lissa, R P; Rajkumar, J S; Sudarsanam, D

    2013-10-01

    Current protocols of islet cell transplantation for the treatment of diabetes mellitus have been hampered by islet availability and allograft rejection. Although bone marrow and subcutaneous adipose tissue stem cells feature their tissue repair efficacy, applicability of stem cells from various sources is being researched to develop a promising therapy for diabetes mellitus. Although omentum fat has emerged as an innovative source of stem cells, the dearth of researches confirming its transdifferentiation potential limits its applicability as a regenerative tool in diabetic therapy. Thus, this work is a maiden attempt to explore the colossal potency of omentum fat-derived stem cells on its lucrative differentiation ability. The plasticity of omentum fat stem cells was substantiated by transdifferentiation into pancreatic islet-like clusters, which was confirmed by dithizone staining and immunocytochemistry for insulin. It was also confirmed by the expression of pancreatic endocrine markers nestin and pancreatic duodenal homeobox 1 (Pdx 1) using Fluorescence-activated cell sorting (FACS), neurogenic 3, islet-1 transcription factor, paired box gene 4, Pdx 1 and insulin using quantitative real-time polymerase chain reaction and through insulin secretion assay. This study revealed the in vitro differentiation potency of omentum fat stem cells into pancreatic islet-like clusters. However, further research pursuits exploring its in vivo endocrine efficacy would make omentum fat stem cells a superior source for β-cell replacement therapy. Copyright © 2013 John Wiley & Sons, Ltd.

  11. Detecting and extracting clusters in atom probe data: A simple, automated method using Voronoi cells

    International Nuclear Information System (INIS)

    Felfer, P.; Ceguerra, A.V.; Ringer, S.P.; Cairney, J.M.

    2015-01-01

    The analysis of the formation of clusters in solid solutions is one of the most common uses of atom probe tomography. Here, we present a method where we use the Voronoi tessellation of the solute atoms and its geometric dual, the Delaunay triangulation to test for spatial/chemical randomness of the solid solution as well as extracting the clusters themselves. We show how the parameters necessary for cluster extraction can be determined automatically, i.e. without user interaction, making it an ideal tool for the screening of datasets and the pre-filtering of structures for other spatial analysis techniques. Since the Voronoi volumes are closely related to atomic concentrations, the parameters resulting from this analysis can also be used for other concentration based methods such as iso-surfaces. - Highlights: • Cluster analysis of atom probe data can be significantly simplified by using the Voronoi cell volumes of the atomic distribution. • Concentration fields are defined on a single atomic basis using Voronoi cells. • All parameters for the analysis are determined by optimizing the separation probability of bulk atoms vs clustered atoms

  12. Detecting and extracting clusters in atom probe data: A simple, automated method using Voronoi cells

    Energy Technology Data Exchange (ETDEWEB)

    Felfer, P., E-mail: peter.felfer@sydney.edu.au [Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006 (Australia); School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, NSW 2006 (Australia); Ceguerra, A.V., E-mail: anna.ceguerra@sydney.edu.au [Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006 (Australia); School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, NSW 2006 (Australia); Ringer, S.P., E-mail: simon.ringer@sydney.edu.au [Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006 (Australia); School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, NSW 2006 (Australia); Cairney, J.M., E-mail: julie.cairney@sydney.edu.au [Australian Centre for Microscopy and Microanalysis, The University of Sydney, NSW 2006 (Australia); School of Aerospace, Mechanical and Mechatronic Engineering, The University of Sydney, NSW 2006 (Australia)

    2015-03-15

    The analysis of the formation of clusters in solid solutions is one of the most common uses of atom probe tomography. Here, we present a method where we use the Voronoi tessellation of the solute atoms and its geometric dual, the Delaunay triangulation to test for spatial/chemical randomness of the solid solution as well as extracting the clusters themselves. We show how the parameters necessary for cluster extraction can be determined automatically, i.e. without user interaction, making it an ideal tool for the screening of datasets and the pre-filtering of structures for other spatial analysis techniques. Since the Voronoi volumes are closely related to atomic concentrations, the parameters resulting from this analysis can also be used for other concentration based methods such as iso-surfaces. - Highlights: • Cluster analysis of atom probe data can be significantly simplified by using the Voronoi cell volumes of the atomic distribution. • Concentration fields are defined on a single atomic basis using Voronoi cells. • All parameters for the analysis are determined by optimizing the separation probability of bulk atoms vs clustered atoms.

  13. Diffusion wave and signal transduction in biological live cells

    OpenAIRE

    Fan, Tian You; Fan, Lei

    2012-01-01

    Transduction of mechanical stimuli into biochemical signals is a fundamental subject for cell physics. In the experiments of FRET signal in cells a wave propagation in nanoscope was observed. We here develop a diffusion wave concept and try to give an explanation to the experimental observation. The theoretical prediction is in good agreement to result of the experiment.

  14. Truly Nonionic Polymer Shells for the Encapsulation of Living Cells

    Science.gov (United States)

    2011-07-04

    successfully been encapsulated using LbL assembly include stem cells, bacteria , bacteria spores, pancreatic islets, and plate- lets.[49–54] The use...cerevisiae YPH501 diploid yeast strain expressing yEGFP (yeast-enhanced GFP) were used for this study.[39] Cells were cultured in synthetic minimal medium

  15. Direct observation of nanomechanical properties of chromatin in living cells

    NARCIS (Netherlands)

    de Vries, Anthony H B; Krenn, Bea E; van Driel, Roel; Subramaniam, Vinod; Kanger, Johannes S

    Precise manipulation of nanometer-sized magnetic particles using magnetic tweezers has yielded insights into the rheology of the cell cytoplasm. We present first results using this approach to study the nanomechanics of the cell nucleus. Using a custom-designed micro-magnetic-tweezers instrument, we

  16. 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.

  17. 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

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

    NARCIS (Netherlands)

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

    2001-01-01

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

  19. Evaluation of reflection interference contrast microscope images of living cells.

    Science.gov (United States)

    Beck, K; Bereiter-Hahn, J

    1981-03-01

    Reflection contrast microscope methods are generally used for studies of those portions of the cell that are turned towards the glass coverslip, to comprehend the structure of the cytoskeleton and the dynamics of cell movement, as well as formation of cell-glass contacts. In incident illumination only reflected light contributes to picture formation. The intensity of which in the case of observation of unstained cells is small because of small refraction differences. To overcome this problem a reflection contrast system was developed by Leitz according to Ploem [49], in which by using contrast preserving measures the reflection becomes prominent in comparison with the lens reflexes. The emerging pictures are a result of interferences of reflections at glass-cell, cell-culture medium and culture medium-cell interfaces. According to Fresnel's equations the reflected intensity depends on the differences of the particular refractive indices and the thickness of the layers, which determine the phase of interfering beams. In idealized systems of thin films the reflected intensity is a measure for their optical constants. Relative reflection measurements from glass-cell areas is comparison with the known glass-medium reflection, can therefore be revealing as far as refraction index, cell-glass distance or cell thickness are concerned. The estimates by Bereiter-Hahn et al. [15] were made in the assumption of vertical illumination neglecting its actual conical shape: the comparison of two Fresnel functions of cytological relevant measurements show - in accordance with Gingell and Todd [24] - that this is only permitted under certain conditions, depending on the required accuracy of the measurements; an incidence angle of about 30 degrees leads to an error of about 10%, an angle of 50 degrees to more than 50%.

  20. rBC2LCN, a new probe for live cell imaging of human pluripotent stem cells.

    Science.gov (United States)

    Onuma, Yasuko; Tateno, Hiroaki; Hirabayashi, Jun; Ito, Yuzuru; Asashima, Makoto

    2013-02-15

    Cell surface biomarkers have been applied to discriminate pluripotent human embryonic stem cells and induced pluripotent stem cells from differentiated cells. Here, we demonstrate that a recombinant lectin probe, rBC2LCN, a new tool for fluorescence-based imaging and flow cytometry analysis of pluripotent stem cells, is an alternative to conventional pluripotent maker antibodies. Live or fixed colonies of both human embryonic stem cells and induced pluripotent stem cells were visualized in culture medium containing fluorescent dye-labeled rBC2LCN. Fluorescent dye-labeled rBC2LCN was also successfully used to separate live pluripotent stem cells from a mixed cell population by flow cytometry. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Peculiarities of living cell response to the external stimuli revealed via quasistatic mode of atomic force microscopy

    Science.gov (United States)

    Khalisov, M. M.; Ankudinov, A. V.; Penniyaynen, V. A.; Timoshenko, T. E.; Timoshchuk, K. I.; Samsonov, M. V.; Shirinsky, V. P.

    2017-10-01

    The technique of atomic force microscopy allows revealing living cell morphology and mechanical properties characterization under physiologically relevant conditions. Here, we review our recent results on living cell reaction to different external influences obtained by this technique. The Bruker PeakForce QNM quasistatic mode was used to study living fibroblasts, erythrocytes, sensory neurons, and endothelial cells.

  2. Control of clustered action potential firing in a mathematical model of entorhinal cortex stellate cells.

    Science.gov (United States)

    Tait, Luke; Wedgwood, Kyle; Tsaneva-Atanasova, Krasimira; Brown, Jon T; Goodfellow, Marc

    2018-04-11

    The entorhinal cortex is a crucial component of our memory and spatial navigation systems and is one of the first areas to be affected in dementias featuring tau pathology, such as Alzheimer's disease and frontotemporal dementia. Electrophysiological recordings from principle cells of medial entorhinal cortex (layer II stellate cells, mEC-SCs) demonstrate a number of key identifying properties including subthreshold oscillations in the theta (4-12 Hz) range and clustered action potential firing. These single cell properties are correlated with network activity such as grid firing and coupling between theta and gamma rhythms, suggesting they are important for spatial memory. As such, experimental models of dementia have revealed disruption of organised dorsoventral gradients in clustered action potential firing. To better understand the mechanisms underpinning these different dynamics, we study a conductance based model of mEC-SCs. We demonstrate that the model, driven by extrinsic noise, can capture quantitative differences in clustered action potential firing patterns recorded from experimental models of tau pathology and healthy animals. The differential equation formulation of our model allows us to perform numerical bifurcation analyses in order to uncover the dynamic mechanisms underlying these patterns. We show that clustered dynamics can be understood as subcritical Hopf/homoclinic bursting in a fast-slow system where the slow sub-system is governed by activation of the persistent sodium current and inactivation of the slow A-type potassium current. In the full system, we demonstrate that clustered firing arises via flip bifurcations as conductance parameters are varied. Our model analyses confirm the experimentally suggested hypothesis that the breakdown of clustered dynamics in disease occurs via increases in AHP conductance. Copyright © 2018. Published by Elsevier Ltd.

  3. Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane.

    Directory of Open Access Journals (Sweden)

    Teruhisa Takagishi

    Full Text Available Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB. DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1, a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step and activation of endogenous PLCγ-1 (second step leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA.

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

    Science.gov (United States)

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

    2016-02-01

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

  5. A phase synchronization clustering algorithm for identifying interesting groups of genes from cell cycle expression data

    Directory of Open Access Journals (Sweden)

    Tcha Hong

    2008-01-01

    Full Text Available Abstract Background The previous studies of genome-wide expression patterns show that a certain percentage of genes are cell cycle regulated. The expression data has been analyzed in a number of different ways to identify cell cycle dependent genes. In this study, we pose the hypothesis that cell cycle dependent genes are considered as oscillating systems with a rhythm, i.e. systems producing response signals with period and frequency. Therefore, we are motivated to apply the theory of multivariate phase synchronization for clustering cell cycle specific genome-wide expression data. Results We propose the strategy to find groups of genes according to the specific biological process by analyzing cell cycle specific gene expression data. To evaluate the propose method, we use the modified Kuramoto model, which is a phase governing equation that provides the long-term dynamics of globally coupled oscillators. With this equation, we simulate two groups of expression signals, and the simulated signals from each group shares their own common rhythm. Then, the simulated expression data are mixed with randomly generated expression data to be used as input data set to the algorithm. Using these simulated expression data, it is shown that the algorithm is able to identify expression signals that are involved in the same oscillating process. We also evaluate the method with yeast cell cycle expression data. It is shown that the output clusters by the proposed algorithm include genes, which are closely associated with each other by sharing significant Gene Ontology terms of biological process and/or having relatively many known biological interactions. Therefore, the evaluation analysis indicates that the method is able to identify expression signals according to the specific biological process. Our evaluation analysis also indicates that some portion of output by the proposed algorithm is not obtainable by the traditional clustering algorithm with

  6. [Natural nanotechnologies in living cell: view on the problem].

    Science.gov (United States)

    Chekman, I S

    2012-12-01

    Literature and research data concerning natural nanotechnology in living systems is summarized in this article. Over millions of years, nature has evolved complex processes controlling the functioning of living organisms. Using the principles of nature to create new tools and technologies is the current direction in the development of "smart" materials and systems. Physiologically active substances (amino acids, neurotransmitters, vitamins, albumin, ATP, fructose, DNA, RNA, fibrinogen, glutathione and others) are nano in size. Biological membranes, ion channels, nanopores, colloidal solutions of the body (blood, interstitial fluid) are nanostructures. The basic principle of nature reads: small tools make great work. In biological materials natural principle of high efficiency and minimum energy consumption is carried out due to the existence of natural nanotechnology that needs more study. In efficient energy-consuming processes during the evolution gradually disappeared and were replaced by more effective. The study and experimental proof of natural nanotechnology will bring a better understanding of the laws of physiological functioning of organs and those of metabolic processes that maintain life. The ability to reproduce biological form with nanoscale precision will find application in tissue engineering, drug delivery, modeling and development of sensory and immune systems, visualization and diagnostic tools. Natural nanotechnology is highly effective, low energy-consuming, causes a rapid flow of vital processes in the body, the ability to quickly response to external positive and negative factors. Not all data presented in this paper is experimentally confirmed, it may be controversial and require further extensive research by specialists of different areas to determine the role of nanotechnology and nanomechanisms in physiological, biochemical, immunological, genetic and other processes in living systems.

  7. Live imaging of Drosophila gonad formation reveals roles for Six4 in regulating germline and somatic cell migration

    Directory of Open Access Journals (Sweden)

    Jarman Andrew P

    2007-05-01

    Full Text Available Abstract Background Movement of cells, either as amoeboid individuals or in organised groups, is a key feature of organ formation. Both modes of migration occur during Drosophila embryonic gonad development, which therefore provides a paradigm for understanding the contribution of these processes to organ morphogenesis. Gonads of Drosophila are formed from three distinct cell types: primordial germ cells (PGCs, somatic gonadal precursors (SGPs, and in males, male-specific somatic gonadal precursors (msSGPs. These originate in distinct locations and migrate to associate in two intermingled clusters which then compact to form the spherical primitive gonads. PGC movements are well studied, but much less is known of the migratory events and other interactions undergone by their somatic partners. These appear to move in organised groups like, for example, lateral line cells in zebra fish or Drosophila ovarian border cells. Results We have used time-lapse fluorescence imaging to characterise gonadal cell behaviour in wild type and mutant embryos. We show that the homeodomain transcription factor Six4 is required for the migration of the PGCs and the msSGPs towards the SGPs. We have identified a likely cause of this in the case of PGCs as we have found that Six4 is required for expression of Hmgcr which codes for HMGCoA reductase and is necessary for attraction of PGCs by SGPs. Six4 affects msSGP migration by a different pathway as these move normally in Hmgcr mutant embryos. Additionally, embryos lacking fully functional Six4 show a novel phenotype in which the SGPs, which originate in distinct clusters, fail to coalesce to form unified gonads. Conclusion Our work establishes the Drosophila gonad as a model system for the analysis of coordinated cell migrations and morphogenesis using live imaging and demonstrates that Six4 is a key regulator of somatic cell function during gonadogenesis. Our data suggest that the initial association of SGP clusters

  8. Embryonic zebrafish primary cell culture for transfection and live cellular and subcellular imaging.

    Science.gov (United States)

    Sassen, Wiebke A; Lehne, Franziska; Russo, Giulio; Wargenau, Sven; Dübel, Stefan; Köster, Reinhard W

    2017-10-01

    Although having great potential for live cell imaging to address numerous cell biological questions with high spatial and temporal resolution, primary cell cultures of zebrafish embryos are not widely used. We present an easy-to-use protocol for preparing primary cell cultures of 2 dpf zebrafish embryos allowing for live cell imaging of fully differentiated cells such as neurons and myocytes. We demonstrate that different cell types can be identified by morphology and expression of transgenic cell type-specific fluorescent reporters and that fluorescent cells can be sorted by flow cytometry to prepare an enriched culture. To facilitate subcellular imaging in live primary cells, we successfully tested a selection of fluorescent vital dyes. Most importantly, we demonstrate that zebrafish primary cells can be transfected efficiently with expression constructs allowing for visualizing subcellular structures with fluorescent marker proteins for time lapse imaging. We propose zebrafish primary cell culture as a versatile tool to address cell biological questions in combination with a powerful in vivo model. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    Griffin, Donald R; Kasko, Andrea M

    2012-08-08

    Hydrogel scaffolds are commonly used as 3D carriers for cells because their properties can be tailored to match natural extracellular matrix. Hydrogels may be used in tissue engineering and regenerative medicine to deliver therapeutic cells to injured or diseased tissue through controlled degradation. Hydrolysis and enzymolysis are the two most common mechanisms employed for hydrogel degradation, but neither allows sequential or staged release of cells. In contrast, photodegradation allows external real-time spatial and temporal control over hydrogel degradation, and allows for staged and sequential release of cells. We synthesized and characterized a series of macromers incorporating photodegradbale ortho-nitrobenzyl (o-NB) groups in the macromer backbone. We formed hydrogels from these macromers via redox polymerization and quantified the apparent rate constants of degradation (kapp) of each via photorheology at 370 nm, 10 mW/cm(2). Decreasing the number of aryl ethers on the o-NB group increases kapp, and changing the functionality from primary to seconday at the benzylic site dramatically increases kapp. Human mesenchymal stem cells (hMSCs) survive encapsulation in the hydrogels (90% viability postencapsulation). By exploiting the differences in reactivity of two different o-NB linkers, we quantitatively demonstrate the biased release of one stem cell population (green-fluoroescent protein expressing hMSCs) over another (red-fluorescent protein expressing hMSCs).

  10. 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.

  11. A novel approach for studying programmed cell death in living plant tissues

    DEFF Research Database (Denmark)

    Mark, Christina

    signaling and the cellular redox environment in aleurone layers undergoing PCD is mainly based on analyses of cell extracts, which do not evaluate the overall cellular redox environment in intact plant cells. Secondly, analyses based on cell extracts are end-point measurements, which are limited by the fact...... insight by determining both the intra- and extracellular reducing capacity in living cells rather than using cell extracts. The reducing capacity of aleurone cells was shown to increase over time in parallel with the increase in cell death. Use of the flavoenzyme inhibitor diphenyleneiodonium chloride...

  12. Live-cell imaging of biothiols via thiol/disulfide exchange to trigger the photoinduced electron transfer of gold-nanodot sensor

    International Nuclear Information System (INIS)

    Liu, Ching-Ping; Wu, Te-Haw; Liu, Chia-Yeh; Lin, Shu-Yi

    2014-01-01

    Highlights: • The ultrasmall size, PAMAM dendrimer-entrapped Au 8 -clusters were synthesized. • Thiol/disulfide exchange with biothiols to release 2-PyT resulted in quenching. • The sensing platform can detect both low and high molecular weight thiols. • Capable of imaging biothiols including protein thiols in living cells. - Abstract: Biothiols have been reported to involve in intracellular redox-homeostasis against oxidative stress. In this study, a highly selective and sensitive fluorescent probe for sensing biothiols is explored by using an ultrasmall gold nanodot (AuND), the dendrimer-entrapped Au 8 -cluster. This strategy relies upon a thiol/disulfide exchange to trigger the fluorescence change through a photoinduced electron transfer (PET) process between the Au 8 -cluster (as an electron donor) and 2-pyridinethiol (2-PyT) (as an electron acceptor) for sensing biothiols. When 2-PyT is released via the cleavage of disulfide bonds by biothiols, the PET process from the Au 8 -cluster to 2-PyT is initiated, resulting in fluorescence quenching. The fluorescence intensity was found to decrease linearly with glutathione (GSH) concentration (0–1500 μM) at physiological relevant levels and the limit of detection for GSH was 15.4 μM. Compared to most nanoparticle-based fluorescent probes that are limited to detect low molecular weight thiols (LMWTs; i.e., GSH and cysteine), the ultrasmall Au 8 -cluster-based probe exhibited less steric hindrance and can be directly applied in selectively and sensitively detecting both LMWTs and high molecular weight thiols (HMWTs; i.e., protein thiols). Based on such sensing platform, the surface-functionalized Au 8 -cluster has significant promise for use as an efficient nanoprobe for intracellular fluorescence imaging of biothiols including protein thiols in living cells whereas other nanoparticle-based fluorescent probes cannot

  13. Live-cell imaging of biothiols via thiol/disulfide exchange to trigger the photoinduced electron transfer of gold-nanodot sensor

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ching-Ping; Wu, Te-Haw; Liu, Chia-Yeh; Lin, Shu-Yi, E-mail: shuyi@nhri.org.tw

    2014-11-07

    Highlights: • The ultrasmall size, PAMAM dendrimer-entrapped Au{sub 8}-clusters were synthesized. • Thiol/disulfide exchange with biothiols to release 2-PyT resulted in quenching. • The sensing platform can detect both low and high molecular weight thiols. • Capable of imaging biothiols including protein thiols in living cells. - Abstract: Biothiols have been reported to involve in intracellular redox-homeostasis against oxidative stress. In this study, a highly selective and sensitive fluorescent probe for sensing biothiols is explored by using an ultrasmall gold nanodot (AuND), the dendrimer-entrapped Au{sub 8}-cluster. This strategy relies upon a thiol/disulfide exchange to trigger the fluorescence change through a photoinduced electron transfer (PET) process between the Au{sub 8}-cluster (as an electron donor) and 2-pyridinethiol (2-PyT) (as an electron acceptor) for sensing biothiols. When 2-PyT is released via the cleavage of disulfide bonds by biothiols, the PET process from the Au{sub 8}-cluster to 2-PyT is initiated, resulting in fluorescence quenching. The fluorescence intensity was found to decrease linearly with glutathione (GSH) concentration (0–1500 μM) at physiological relevant levels and the limit of detection for GSH was 15.4 μM. Compared to most nanoparticle-based fluorescent probes that are limited to detect low molecular weight thiols (LMWTs; i.e., GSH and cysteine), the ultrasmall Au{sub 8}-cluster-based probe exhibited less steric hindrance and can be directly applied in selectively and sensitively detecting both LMWTs and high molecular weight thiols (HMWTs; i.e., protein thiols). Based on such sensing platform, the surface-functionalized Au{sub 8}-cluster has significant promise for use as an efficient nanoprobe for intracellular fluorescence imaging of biothiols including protein thiols in living cells whereas other nanoparticle-based fluorescent probes cannot.

  14. Monitoring the cytoskeletal EGF response in live gastric carcinoma cells.

    Directory of Open Access Journals (Sweden)

    Marco Felkl

    Full Text Available Altered cell motility is considered to be a key factor in determining tumor invasion and metastasis. Epidermal growth factor (EGF signaling has been implicated in this process by affecting cytoskeletal organization and dynamics in multiple ways. To sort the temporal and spatial regulation of EGF-dependent cytoskeletal re-organization in relation to a cell's motile behavior time-lapse microscopy was performed on EGF-responsive gastric carcinoma-derived MKN1 cells co-expressing different fluorescently labeled cytoskeletal filaments and focal adhesion components in various combinations. The experiments showed that EGF almost instantaneously induces a considerable increase in membrane ruffling and lamellipodial activity that can be inhibited by Cetuximab EGF receptor antibodies and is not elicited in non-responsive gastric carcinoma Hs746T cells. The transient cell extensions are rich in actin but lack microtubules and keratin intermediate filaments. We show that this EGF-induced increase in membrane motility can be measured by a simple image processing routine. Microtubule plus-ends subsequently invade growing cell extensions, which start to accumulate focal complexes at the lamellipodium-lamellum junction. Such paxillin-positive complexes mature into focal adhesions by tyrosine phosphorylation and recruitment of zyxin. These adhesions then serve as nucleation sites for keratin filaments which are used to enlarge the neighboring peripheral keratin network. Focal adhesions are either disassembled or give rise to stable zyxin-rich fibrillar adhesions which disassemble in the presence of EGF to support formation of new focal adhesion sites in the cell periphery. Taken together the results serve as a basis for modeling the early cytoskeletal EGF response as a tightly coordinated and step-wise process which is relevant for the prediction of the effectiveness of anti-EGF receptor-based tumor therapy.

  15. 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.

  16. 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....

  17. Stretchable living materials and devices with hydrogel-elastomer hybrids hosting programmed cells.

    Science.gov (United States)

    Liu, Xinyue; Tang, Tzu-Chieh; Tham, Eléonore; Yuk, Hyunwoo; Lin, Shaoting; Lu, Timothy K; Zhao, Xuanhe

    2017-02-28

    Living systems, such as bacteria, yeasts, and mammalian cells, can be genetically programmed with synthetic circuits that execute sensing, computing, memory, and response functions. Integrating these functional living components into materials and devices will provide powerful tools for scientific research and enable new technological applications. However, it has been a grand challenge to maintain the viability, functionality, and safety of living components in freestanding materials and devices, which frequently undergo deformations during applications. Here, we report the design of a set of living materials and devices based on stretchable, robust, and biocompatible hydrogel-elastomer hybrids that host various types of genetically engineered bacterial cells. The hydrogel provides sustainable supplies of water and nutrients, and the elastomer is air-permeable, maintaining long-term viability and functionality of the encapsulated cells. Communication between different bacterial strains and with the environment is achieved via diffusion of molecules in the hydrogel. The high stretchability and robustness of the hydrogel-elastomer hybrids prevent leakage of cells from the living materials and devices, even under large deformations. We show functions and applications of stretchable living sensors that are responsive to multiple chemicals in a variety of form factors, including skin patches and gloves-based sensors. We further develop a quantitative model that couples transportation of signaling molecules and cellular response to aid the design of future living materials and devices.

  18. 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.

  19. The Beta Cell in Its Cluster: Stochastic Graphs of Beta Cell Connectivity in the Islets of Langerhans.

    Directory of Open Access Journals (Sweden)

    Deborah A Striegel

    2015-08-01

    Full Text Available Pancreatic islets of Langerhans consist of endocrine cells, primarily α, β and δ cells, which secrete glucagon, insulin, and somatostatin, respectively, to regulate plasma glucose. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Due to the central functional significance of this local connectivity in the placement of β cells in an islet, it is important to characterize it quantitatively. However, quantification of the seemingly stochastic cytoarchitecture of β cells in an islet requires mathematical methods that can capture topological connectivity in the entire β-cell population in an islet. Graph theory provides such a framework. Using large-scale imaging data for thousands of islets containing hundreds of thousands of cells in human organ donor pancreata, we show that quantitative graph characteristics differ between control and type 2 diabetic islets. Further insight into the processes that shape and maintain this architecture is obtained by formulating a stochastic theory of β-cell rearrangement in whole islets, just as the normal equilibrium distribution of the Ornstein-Uhlenbeck process can be viewed as the result of the interplay between a random walk and a linear restoring force. Requiring that rearrangements maintain the observed quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that β-cell rearrangement is dependent on its connectivity in order to maintain an optimal cluster size in both normal and T2D islets.

  20. Dynamics of Ras Complexes Observed in Living Cells

    Directory of Open Access Journals (Sweden)

    Xiangyong Li

    2012-07-01

    Full Text Available K-Ras works as a switch in many important intracellular signaling pathways and plays important roles in cell growth, proliferation, differentiation and carcinogenesis. For signal transduction from K-Ras to Raf1, the best-characterized effector of K-Ras, the general view is that Ras recruits Raf1 from the cytoplasm to the cell membrane. To elucidate this process, we constructed a series of fusion proteins (including Raf1 and K-Ras fused with either fluorescent proteins or fluorescent protein fragments to compare subcellular localizations of these proteins. Bimolecular fluorescence complementation (BiFC and a co-transfection system were used. In the BiFC system, the K-Ras/Raf1 complexes were mainly located in the cell membrane, while the Raf1 control was uniformly distributed in the cytoplasm. However, the complexes of Raf1 and K-RasC185S, a K-Ras mutant which loses membrane-localization, were also able to accumulate in the cell membrane. In contrast, an apparent cytosolic distribution pattern was observed in cells co-transfected with mcerulean-Raf1 and EGFP-K-RasC185S, suggesting that the membrane localization of K-Ras/Raf1 complexes is not entirely dependent on K-Ras, and that other factors, such as the irreversible conformation formed between K-Ras and Raf1 may play a role. This study sheds light on the interaction between K-Ras and Raf1 and provides a practical method to elucidate the mechanism underlying K-Ras and Raf1 binding to the cell membrane.

  1. High-speed atomic force microscopy imaging of live mammalian cells.

    Science.gov (United States)

    Shibata, Mikihiro; Watanabe, Hiroki; Uchihashi, Takayuki; Ando, Toshio; Yasuda, Ryohei

    2017-01-01

    Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons.

  2. Building cells for quantitative, live-cell analyses of collective motor protein functions.

    Science.gov (United States)

    Kumar, Eric A; Tsao, David; Radhakrishnan, Anand; Diehl, Michael

    2015-01-01

    Examining the collective mechanical behaviors of interacting cytoskeletal motors has become increasingly important to dissecting the complex and multifaceted mechanisms that regulate the transport and trafficking of materials in cells. Although studying these processes in living cells has been challenging, the development of new Synthetic Biology techniques has opened unique opportunities to both manipulate and probe how these motors function in groups as they navigate the native cytoskeleton. Here, we describe an approach to engineer mammalian cells for a new class of inducible cargo motility assays that utilize drug-dependent protein dimerization switches to regulate motor-cargo coupling and transport. Our adaptations provide genetic-level control over the densities of motor proteins coupled to, as well as the sizes of endogenous vesicular cargos in these assays. By allowing the examination of transport responses to changes in motor density and cargo size-dependent viscous drag force, such control can enable quantitative comparisons of mechanistic distinctions between the collective behaviors of different types of processive cytoskeletal motors. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Synthesis and Structure-Activity Relationship of Griseofulvin Analogues as Inhibitors of Centrosomal Clustering in Cancer Cells

    DEFF Research Database (Denmark)

    Rønnest, Mads Holger; Rebacz, Blanka; Markworth, Lene

    2009-01-01

    Griseofulvin was identified as an inhibitor of centrosomal clustering in a recently developed assay. Centrosomal clustering is an important cellular event that enables bipolar mitosis for cancer cell lines harboring supernumerary centrosomes. We report herein the synthesis and SAR of 34 griseoful......Griseofulvin was identified as an inhibitor of centrosomal clustering in a recently developed assay. Centrosomal clustering is an important cellular event that enables bipolar mitosis for cancer cell lines harboring supernumerary centrosomes. We report herein the synthesis and SAR of 34...

  4. Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells.

    Science.gov (United States)

    Kelbauskas, Laimonas; Shetty, Rishabh; Cao, Bin; Wang, Kuo-Chen; Smith, Dean; Wang, Hong; Chao, Shi-Hui; Gangaraju, Sandhya; Ashcroft, Brian; Kritzer, Margaret; Glenn, Honor; Johnson, Roger H; Meldrum, Deirdre R

    2017-12-01

    Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field.

  5. 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…

  6. 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.

  7. A sensor for quantification of macromolecular crowding in living cells

    NARCIS (Netherlands)

    Boersma, Arnold J; Zuhorn, Inge S; Poolman, Bert

    Macromolecular crowding in cells influences processes such as folding, association and diffusion of proteins and polynucleic acids. Direct spatiotemporal readout of crowding would be a powerful approach for unraveling the structure of the cytoplasm and determining the impact of excluded volume on

  8. 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

    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...

  9. 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

  10. 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.

  11. 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.

  12. Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

    International Nuclear Information System (INIS)

    Mukai, Atsushi; Kurisaki, Tomohiro; Sato, Satoshi B.; Kobayashi, Toshihide; Kondoh, Gen; Hashimoto, Naohiro

    2009-01-01

    Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, β-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

  13. Dynamic clustering and dispersion of lipid rafts contribute to fusion competence of myogenic cells

    Energy Technology Data Exchange (ETDEWEB)

    Mukai, Atsushi [Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Oobu, Aichi 474-8522 (Japan); Kurisaki, Tomohiro [Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507 (Japan); Sato, Satoshi B. [Research Center for Low Temperature and Material Sciences, Kyoto University, Yoshida-honmachi, Kyoto 606-8501 (Japan); Kobayashi, Toshihide [Lipid Biology Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama 351-0198 (Japan); Kondoh, Gen [Laboratory of Animal Experiments for Regeneration, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507 (Japan); Hashimoto, Naohiro, E-mail: nao@nils.go.jp [Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 36-3 Gengo, Morioka, Oobu, Aichi 474-8522 (Japan)

    2009-10-15

    Recent research indicates that the leading edge of lamellipodia of myogenic cells (myoblasts and myotubes) contains presumptive fusion sites, yet the mechanisms that render the plasma membrane fusion-competent remain largely unknown. Here we show that dynamic clustering and dispersion of lipid rafts contribute to both cell adhesion and plasma membrane union during myogenic cell fusion. Adhesion-complex proteins including M-cadherin, {beta}-catenin, and p120-catenin accumulated at the leading edge of lamellipodia, which contains the presumptive fusion sites of the plasma membrane, in a lipid raft-dependent fashion prior to cell contact. In addition, disruption of lipid rafts by cholesterol depletion directly prevented the membrane union of myogenic cell fusion. Time-lapse recording showed that lipid rafts were laterally dispersed from the center of the lamellipodia prior to membrane fusion. Adhesion proteins that had accumulated at lipid rafts were also removed from the presumptive fusion sites when lipid rafts were laterally dispersed. The resultant lipid raft- and adhesion complex-free area at the leading edge fused with the opposing plasma membrane. These results demonstrate a key role for dynamic clustering/dispersion of lipid rafts in establishing fusion-competent sites of the myogenic cell membrane, providing a novel mechanistic insight into the regulation of myogenic cell fusion.

  14. Live cell near-field optical imaging and voltage sensing with ultrasensitive force control.

    Science.gov (United States)

    Brahami, Aaron; Levy, Hadas; Zlotkin-Rivkin, Efrat; Melamed-Book, Naomi; Tal, Nataly; Lev, Dmitry; Yeshua, Talia; Fedosyeyev, Oleg; Aroeti, Benjamin; Lewis, Aaron

    2017-05-29

    Force controlled optical imaging of membranes of living cells is demonstrated. Such imaging has been extended to image membrane potential changes to demonstrate that live cell imaging has been achieved. To accomplish this advance, limitations inherent in atomic force microscopy (AFM) since its inception in 1986 [G. Binnig, C. F. Quate, and C. Gerber, "Atomic Force Microscope," Phys. Rev. Lett. 56, 930-933 (1986).] had to be overcome. The advances allow for live cell imaging of a whole genre of functional biological imaging with stiff (1-10N/m) scanned probe imaging cantilevers. Even topographic imaging of fine cell protrusions, such as microvilli, has been accomplished with such cantilevers. Similar topographic imaging has only recently been demonstrated with the standard soft (0.05N/m) cantilevers that are generally required for live cell imaging. The progress reported here demonstrates both ultrasensitive AFM (~100pN), capable of topographic imaging of even microvilli protruding from cell membranes and new functional applications that should have a significant impact on optical and other approaches in biological imaging of living systems and ultrasoft materials.

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

    Science.gov (United States)

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

    2011-01-01

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

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

    DEFF Research Database (Denmark)

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

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

  17. 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

  18. 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.

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

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba

    2013-01-01

    High-sensitive and high-affinity methods to measure gene expression inside living cells have proven to be invaluable in regards to understanding fundamental processes such as cell differentiation, reprogramming, regeneration and cancer genesis. One tool for transcription visualization on single...

  20. 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

  1. 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.

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

    Science.gov (United States)

    Ranjit, Suman; Lanzano, Luca; Gratton, Enrico

    2014-12-16

    Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data are acquired by scanning each frame once and then scanning the image multiple times. The total time of data acquisition needed for this method is much shorter than the time required for traditional FCS analysis at each pixel. However, at a single pixel, the acquired intensity time sequence is short; requiring nonconventional analysis of the correlation function to extract information about the diffusion. These correlation data have been analyzed using the phasor approach, a fit-free method that was originally developed for analysis of FLIM images. Analysis using this method results in an estimation of the average diffusion coefficient of the fluorescent species at each pixel of an image, and thus, a detailed diffusion map of the cell can be created. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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)

  10. Live-cell imaging of budding yeast telomerase RNA and TERRA.

    Science.gov (United States)

    Laprade, Hadrien; Lalonde, Maxime; Guérit, David; Chartrand, Pascal

    2017-02-01

    In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation. Here, we present detailed protocols for live-cell imaging of the Saccharomyces cerevisiae telomerase RNA subunit, called TLC1, and also of the non-coding telomeric repeat-containing RNA TERRA. We describe the approach used for genomic integration of MS2 stem-loops in these transcripts, and provide information for optimal live-cell imaging of these non-coding RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. 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.

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

    DEFF Research Database (Denmark)

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

    be considered. We have therefore developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion with atomic force microscopy (AFM).[1] A single-cell probe was readily made by picking up a bacterial cell from a glass surface using a tipless AFM cantilever coated...... with a commercial cell adhesive CellTakTM. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, mica). Attachment to the cantilever was stable during the 2 h of AFM force measurements, and viability was confirmed by Live....../Dead fluorescence staining at the end of each experiment. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time of contact. The single-cell probe offers control of the cell immobilization, thus holds advantages over the commonly used multi-cell probes where...

  13. The cost-effectiveness of a new disease management model for frail elderly living in homes for the elderly, design of a cluster randomized controlled clinical trial

    OpenAIRE

    Ribbe Miel W; Frijters Dinnus H; van Hout Hein PJ; Boorsma Marijke; Nijpels Giel

    2008-01-01

    Abstract Background The objective of this article is to describe the design of a study to evaluate the clinical and economic effects of a Disease Management model on functional health, quality of care and quality of life of persons living in homes for the elderly. Methods This study concerns a cluster randomized controlled clinical trial among five intervention homes and five usual care homes in the North-West of the Netherlands with a total of over 500 residents. All persons who are not term...

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

    Science.gov (United States)

    Azzarello, Antonino; Hoest, Thomas; Hay-Schmidt, Anders; Mikkelsen, Anne Lis

    2017-06-01

    This study included 238 good morphology blastocysts, which were transferred after vitrification-warming to 152 women by single blastocyst transfer in Holbæk Fertility Clinic, Denmark. Time-lapse recordings of transferred good morphology blastocysts were reassessed to recognize every abnormal cell division (ACD) from the 1st to the 4th cell cycle. ACDs were distinguished as failed cell divisions and multi-cell divisions. ACDs were recognized in 37.0% (no. 88/238) of good morphology blastocysts that were vitrified-warmed and transferred in our clinic. Good morphology blastocysts with ACDs showed 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: 18.5%; 4th: 18.1%). More blastocysts presented failed cell divisions (no. 95) than multi-cell divisions (no. 14). Live births were achieved from blastocysts showing multi-cell divisions at any cell cycle and failed cell divisions from the 2nd cell cycle. Analyses of the subgroup of first blastocyst transferred to each patient showed similar to results. In conclusion, good morphology blastocysts presenting ACDs can result in live birth although lower compared to blastocysts with solely regular cell division. Pre-implantation embryos in vitro may undergo self-selection or correcting processes. This supports the transfer of blastocysts instead of cleavage stage embryos, giving first priority to blastocyst showing solely regular cell divisions, and giving second priority to blastocysts presenting ACDs at any cell cycle. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  15. 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.

  16. 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.

  17. Live imaging reveals the progenitors and cell dynamics of limb regeneration.

    Science.gov (United States)

    Alwes, Frederike; Enjolras, Camille; Averof, Michalis

    2016-10-25

    Regeneration is a complex and dynamic process, mobilizing diverse cell types and remodelling tissues over long time periods. Tracking cell fate and behaviour during regeneration in active adult animals is especially challenging. Here, we establish continuous live imaging of leg regeneration at single-cell resolution in the crustacean Parhyale hawaiensis . By live recordings encompassing the first 4-5 days after amputation, we capture the cellular events that contribute to wound closure and morphogenesis of regenerating legs with unprecedented resolution and temporal detail. Using these recordings we are able to track cell lineages, to generate fate maps of the blastema and to identify the progenitors of regenerated epidermis. We find that there are no specialized stem cells for the epidermis. Most epidermal cells in the distal part of the leg stump proliferate, acquire new positional values and contribute to new segments in the regenerating leg.

  18. Ultrasensitive Proteome Profiling for 100 Living Cells by Direct Cell Injection, Online Digestion and Nano-LC-MS/MS Analysis.

    Science.gov (United States)

    Chen, Qi; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin

    2015-07-07

    Single-cell proteome analysis has always been an exciting goal because it provides crucial information about cellular heterogeneity and dynamic change. Here we presented an integrated proteome analysis device (iPAD) for 100 living cells (iPAD-100) that might be suitable for single-cell analysis. Once cells were cultured, the iPAD-100 could be applied to inject 100 living cells, to transform the living cells into peptides, and to produce protein identification results with total automation. Due to the major obstacle for detection limit of mass spectrometry, we applied the iPAD-100 to analyze the proteome of 100 cells. In total, 813 proteins were identified in a DLD-cell proteome by three duplicate runs. Gene Ontology analysis revealed that proteins from different cellular compartments were well-represented, including membrane proteins. The iPAD-100 greatly simplified the sampling process, reduced sample loss, and prevented contamination. As a result, proteins whose copy numbers were lower than 1000 were identified from 100-cell samples with the iPAD-100, showing that a detection limit of 200 zmol was achieved. With increased sensitivity of mass spectrometry, the iPAD-100 may be able to reveal bountiful proteome information from a single cell in the near future.

  19. 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.

  20. Active invasion of bacteria into living fungal cells

    Science.gov (United States)

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

    2014-01-01

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

  1. 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

  2. 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%).

  3. 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.

  4. 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

  5. Size and Dynamics of Caveolae Studied Using Nanoparticles in Living Endothelial Cells

    OpenAIRE

    Wang, Zhenjia; Tiruppathi, Chinnaswamy; Minshall, Richard D.; Malik, Asrar B.

    2009-01-01

    Caveolae are plasma membrane invaginations prominent in all endothelial cells lining blood vessels. Caveolae characteristically bud to form free cytoplasmic vesicles capable of transporting carrier proteins such as albumin through the cell. However, caveolae size distribution and dynamics in living endothelial cells and ability of caveolae to internalize nanoparticles are not well understood. We demonstrate here the design of a dual-color nanoparticle pair to measure non-invasively caveolae s...

  6. Cross-linked glucose oxidase clusters for biofuel cell anode catalysts

    International Nuclear Information System (INIS)

    Dudzik, Jonathan; Audette, Gerald F; Chang, Wen-Chi; Kannan, A M; Filipek, Slawomir; Viswanathan, Sowmya; Li, Pingzuo; Renugopalakrishnan, V

    2013-01-01

    The efficient localization of increased levels of active enzymes onto conducting scaffolds is important for the development of enzyme-based biofuel cells. Cross-linked enzyme clusters (CEC) of glucose oxidase (GOx) constrained to functionalized carbon nanotubes (CEC-CNTs) were generated in order to evaluate the potential of using CECs for developing GOx-based bioanodes functioning via direct electron transfer from the GOx active site to the CNT scaffold. CEC-CNTs generated from several weight-to-weight ratios of GOx:CNT were examined for comparable catalytic activity to free GOx into the solution, with CEC-CNTs generated from a 100% GOx solution displaying the greatest enzymatic activity. Scanning transmission electron microscopic analysis of CEC-CNTs generated from 100% GOx to CNT (wt/wt) ratios revealed that CEC clusters of ∼78 µm 2 localized to the CNT surface. Electrochemical analysis indicates that the enzyme is engaged in direct electron transfer, and biofuel cells generated using GOx CEC-CNT bioanodes were observed to have a peak power density of ∼180 µW cm −2 . These data indicate that the generation of nano-to-micro-sized active enzyme clusters is an attractive option for the design of enzyme-specific biofuel cell powered implantable devices. (paper)

  7. Iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery

    Science.gov (United States)

    Maio, Nunziata; Rouault, Tracey. A.

    2014-01-01

    Iron-sulfur (Fe-S) clusters are ancient, ubiquitous cofactors composed of iron and inorganic sulfur. The combination of the chemical reactivity of iron and sulfur, together with many variations of cluster composition, oxidation states and protein environments, enables Fe-S clusters to participate in numerous biological processes. Fe-S clusters are essential to redox catalysis in nitrogen fixation, mitochondrial respiration and photosynthesis, to regulatory sensing in key metabolic pathways (i. e. cellular iron homeostasis and oxidative stress response), and to the replication and maintenance of the nuclear genome. Fe-S cluster biogenesis is a multistep process that involves a complex sequence of catalyzed protein- protein interactions and coupled conformational changes between the components of several dedicated multimeric complexes. Intensive studies of the assembly process have clarified key points in the biogenesis of Fe-S proteins. However several critical questions still remain, such as: what is the role of frataxin? Why do some defects of Fe-S cluster biogenesis cause mitochondrial iron overload? How are specific Fe-S recipient proteins recognized in the process of Fe-S transfer? This review focuses on the basic steps of Fe-S cluster biogenesis, drawing attention to recent advances achieved on the identification of molecular features that guide selection of specific subsets of nascent Fe-S recipients by the cochaperone HSC20. Additionally, it outlines the distinctive phenotypes of human diseases due to mutations in the components of the basic pathway. PMID:25245479

  8. 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.

  9. Human melanocytes form a PAX3-expressing melanocyte cluster on Matrigel by the cell migration process.

    Science.gov (United States)

    Choi, Hyunjung; Jin, Sun Hee; Han, Mi Hwa; Lee, Jinyoung; Ahn, Seyeon; Seong, Minjeong; Choi, Hyun; Han, Jiyeon; Cho, Eun-Gyung; Lee, Tae Ryong; Noh, Minsoo

    2014-10-01

    The interactions between human epidermal melanocytes and their cellular microenvironment are important in the regulation of human melanocyte functions or in their malignant transformation into melanoma. Although the basement membrane extracellular matrix (BM-ECM) is one of major melanocyte microenvironments, the effects of BM-ECM on the human melanocyte functions are not fully explained at a molecular level. This study was aimed to characterize the molecular and cellular interactions between normal human melanocytes (NHMs) and BM-ECM. We investigated cell culture models of normal human melanocytes or melanoma cells on three-dimensional (3D) Matrigel to understand the roles of the basement membrane microenvironment in human melanocyte functions. Melanogenesis and melanobast biomarker expression in both primary human melanocytes and melanoma cells on 3D Matrigel were evaluated. We found that NHMs migrated and formed reversible paired box 3 (PAX3) expressing cell clusters on three-dimensional (3D) Matrigel. The melanogenesis was significantly decreased in the PAX3 expressing cell cluster. The expression profile of PAX3, SOX10, and MITF in the melanocyte cluster on 3D Matrigel was similar to that of melanoblasts. Interestingly, PAX3 and SOX10 showed an inverse expression profile in NHMs, whereas the inverse expression pattern of PAX3 and SOX10 was disrupted in melanoma MNT1 and WM266-4 cells. The human melanocyte culture on 3D Matrigel provides an alternative model system to study functions of human melanoblasts. In addition, this system will contribute to the elucidation of PAX3-related tumorigenic mechanisms to understand human melanoma. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  10. 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.

  11. 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.

  12. DNA-coated AFM cantilevers for the investigation of cell adhesion and the patterning of live cells

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao, Sonny C.; Crow, Ailey K.; Lam, Wilbur A.; Bertozzi, Carolyn R.; Fletcher, Daniel A.; Francis, Matthew B.

    2008-08-01

    Measurement of receptor adhesion strength requires the precise manipulation of single cells on a contact surface. To attach live cells to a moveable probe, DNA sequences complementary to strands displayed on the plasma membrane are introduced onto AFM cantilevers (see picture, bp=base pairs). The strength of the resulting linkages can be tuned by varying the length of DNA strands, allowing for controlled transport of the cells.

  13. 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...

  14. 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

    division (ACD) from the 1st to the 4th cell cycle. ACDs were distinguished as failed cell divisions and multi-cell divisions. ACDs were recognized in 37.0% (no. 88/238) of good morphology blastocysts that were vitrified-warmed and transferred in our clinic. Good morphology blastocysts with ACDs showed...... 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......: 18.5%; 4th: 18.1%). More blastocysts presented failed cell divisions (no. 95) than multi-cell divisions (no. 14). Live births were achieved from blastocysts showing multi-cell divisions at any cell cycle and failed cell divisions from the 2nd cell cycle. Analyses of the subgroup of first blastocyst...

  15. 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

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

    Science.gov (United States)

    Jiang, Xiqian; Yu, Yong; Chen, Jianwei; Zhao, Mingkun; Chen, Hui; Song, Xianzhou; Matzuk, Alexander J; Carroll, Shaina L; Tan, Xiao; Sizovs, Antons; Cheng, Ninghui; Wang, Meng C; Wang, Jin

    2015-03-20

    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 field of cell biology and has become an indispensable tool in current biological studies. In order to minimize the disturbance to the biological system in live cell imaging, the probe concentration needs to be significantly lower than the analyte concentration. Because of this, any irreversible reaction-based GSH probe can only provide qualitative results within a short reaction time and will exhibit maximum response regardless of the GSH concentration if the reaction is completed. A reversible reaction-based probe with an appropriate equilibrium constant allows measurement of an analyte at much higher concentrations and, thus, is a prerequisite for GSH quantification inside cells. In this contribution, we report the first fluorescent probe-ThiolQuant Green (TQ Green)-for quantitative imaging of GSH in live cells. Due to the reversible nature of the reaction between the probe and GSH, we are able to quantify mM concentrations of GSH with TQ Green concentrations as low as 20 nM. Furthermore, the GSH concentrations measured using TQ Green in 3T3-L1, HeLa, HepG2, PANC-1, and PANC-28 cells are reproducible and well correlated with the values obtained from cell lysates. TQ Green imaging can also resolve the changes in GSH concentration in PANC-1 cells upon diethylmaleate (DEM) treatment. In addition, TQ Green can be conveniently applied in fluorescence activated cell sorting (FACS) to measure GSH level changes. Through this study, we not only demonstrate the importance of reaction reversibility in designing quantitative reaction-based fluorescent probes but also provide a practical tool to facilitate redox biology studies.

  17. Correlation of live-cell imaging with volume scanning electron microscopy.

    Science.gov (United States)

    Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

    2017-01-01

    Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Liposome-Coupled Peptides Induce Long-Lived Memory CD8+ T Cells Without CD4+ T Cells

    OpenAIRE

    Taneichi, Maiko; Tanaka, Yuriko; Kakiuchi, Terutaka; Uchida, Tetsuya

    2010-01-01

    CD8(+) T cells provide broad immunity to viruses, because they are able to recognize all types of viral proteins. Therefore, the development of vaccines capable of inducing long-lived memory CD8(+) T cells is desired to prevent diseases, especially those for which no vaccines currently exist. However, in designing CD8(+) T cell vaccines, the role of CD4(+) T cells in the induction and maintenance of memory CD8(+) T cells remains uncertain. In the present study, the necessity or not of CD4(+) ...

  19. 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.

  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. 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.

  2. 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.

  3. 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

  4. Live cell X-ray imaging of autophagic vacuoles formation and chromatin dynamics in fission yeast.

    Science.gov (United States)

    Strelnikova, Natalja; Sauter, Nora; Guizar-Sicairos, Manuel; Göllner, Michael; Diaz, Ana; Delivani, Petrina; Chacón, Mariola; Tolić, Iva M; Zaburdaev, Vasily; Pfohl, Thomas

    2017-10-23

    Seeing physiological processes at the nanoscale in living organisms without labeling is an ultimate goal in life sciences. Using X-ray ptychography, we explored in situ the dynamics of unstained, living fission yeast Schizosaccharomyces pombe cells in natural, aqueous environment at the nanoscale. In contrast to previous X-ray imaging studies on biological matter, in this work the eukaryotic cells were alive even after several ptychographic X-ray scans, which allowed us to visualize the chromatin motion as well as the autophagic cell death induced by the ionizing radiation. The accumulated radiation of the sequential scans allowed for the determination of a characteristic dose of autophagic vacuole formation and the lethal dose for fission yeast. The presented results demonstrate a practical method that opens another way of looking at living biological specimens and processes in a time-resolved label-free setting.

  5. Quantum Dots for Live Cell and In Vivo Imaging

    Directory of Open Access Journals (Sweden)

    Jason R. E. Shepard

    2009-02-01

    Full Text Available In the past few decades, technology has made immeasurable strides to enable visualization, identification, and quantitation in biological systems. Many of these technological advancements are occurring on the nanometer scale, where multiple scientific disciplines are combining to create new materials with enhanced properties. The integration of inorganic synthetic methods with a size reduction to the nano-scale has lead to the creation of a new class of optical reporters, called quantum dots. These semiconductor quantum dot nanocrystals have emerged as an alternative to organic dyes and fluorescent proteins, and are brighter and more stable against photobleaching than standard fluorescent indicators. Quantum dots have tunable optical properties that have proved useful in a wide range of applications from multiplexed analysis such as DNA detection and cell sorting and tracking, to most recently demonstrating promise for in vivo imaging and diagnostics. This review provides an in-depth discussion of past, present, and future trends in quantum dot use with an emphasis on in vivo imaging and its related applications.

  6. 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

  7. 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.

  8. 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.

  9. A critical assessment of geographic clusters of breast and lung cancer incidences among residents living near the Tittabawassee and Saginaw Rivers, Michigan, USA.

    Science.gov (United States)

    Guajardo, Olga A; Oyana, Tonny J

    2009-01-01

    To assess previously determined geographic clusters of breast and lung cancer incidences among residents living near the Tittabawassee and Saginaw Rivers, Michigan, using a new set of environmental factors. Breast and lung cancer data were acquired from the Michigan Department of Community Health, along with point source pollution data from the U.S. Environmental Protection Agency. The datasets were used to determine whether there is a spatial association between disease risk and environmental contamination. GIS and spatial techniques were combined with statistical analysis to investigate local risk of breast and lung cancer. The study suggests that neighborhoods in close proximity to the river were associated with a high risk of breast cancer, while increased risk of lung cancer was detected among neighborhoods in close proximity to point source pollution and major highways. Statistically significant (P clusters of cancer incidences were observed among residents living near the rivers. These findings are useful to researchers and governmental agencies for risk assessment, regulation, and control of environmental contamination in the floodplains.

  10. A Critical Assessment of Geographic Clusters of Breast and Lung Cancer Incidences among Residents Living near the Tittabawassee and Saginaw Rivers, Michigan, USA

    International Nuclear Information System (INIS)

    Guajardo, O.A.; Oyana, T.J.

    2010-01-01

    Objectives. To assess previously determined geographic clusters of breast and lung cancer incidences among residents living near the Tittabawassee and Saginaw Rivers, Michigan, using a new set of environmental factors. Materials and Methods. Breast and lung cancer data were acquired from the Michigan Department of Community Health, along with point source pollution data from the U.S. Environmental Protection Agency. The datasets were used to determine whether there is a spatial association between disease risk and environmental contamination. GIS and spatial techniques were combined with statistical analysis to investigate local risk of breast and lung cancer. Results and Conclusion. The study suggests that neighborhoods in close proximity to the river were associated with a high risk of breast cancer, while increased risk of lung cancer was detected among neighborhoods in close proximity to point source pollution and major highways. Statistically significant (P=.001) clusters of cancer incidences were observed among residents living near the rivers. These findings are useful to researchers and governmental agencies for risk assessment, regulation, and control of environmental contamination in the flood plains.

  11. A Critical Assessment of Geographic Clusters of Breast and Lung Cancer Incidences among Residents Living near the Tittabawassee and Saginaw Rivers, Michigan, USA

    Directory of Open Access Journals (Sweden)

    Olga A. Guajardo

    2009-01-01

    Full Text Available Objectives. To assess previously determined geographic clusters of breast and lung cancer incidences among residents living near the Tittabawassee and Saginaw Rivers, Michigan, using a new set of environmental factors. Materials and Methods. Breast and lung cancer data were acquired from the Michigan Department of Community Health, along with point source pollution data from the U.S. Environmental Protection Agency. The datasets were used to determine whether there is a spatial association between disease risk and environmental contamination. GIS and spatial techniques were combined with statistical analysis to investigate local risk of breast and lung cancer. Results and Conclusion. The study suggests that neighborhoods in close proximity to the river were associated with a high risk of breast cancer, while increased risk of lung cancer was detected among neighborhoods in close proximity to point source pollution and major highways. Statistically significant (P≤.001 clusters of cancer incidences were observed among residents living near the rivers. These findings are useful to researchers and governmental agencies for risk assessment, regulation, and control of environmental contamination in the floodplains.

  12. 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

  13. 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.

  14. 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.

  15. 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...

  16. 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.

  17. A Novel Automated High-Content Analysis Workflow Capturing Cell Population Dynamics from Induced Pluripotent Stem Cell Live Imaging Data.

    Science.gov (United States)

    Kerz, Maximilian; Folarin, Amos; Meleckyte, Ruta; Watt, Fiona M; Dobson, Richard J; Danovi, Davide

    2016-10-01

    Most image analysis pipelines rely on multiple channels per image with subcellular reference points for cell segmentation. Single-channel phase-contrast images are often problematic, especially for cells with unfavorable morphology, such as induced pluripotent stem cells (iPSCs). Live imaging poses a further challenge, because of the introduction of the dimension of time. Evaluations cannot be easily integrated with other biological data sets including analysis of endpoint images. Here, we present a workflow that incorporates a novel CellProfiler-based image analysis pipeline enabling segmentation of single-channel images with a robust R-based software solution to reduce the dimension of time to a single data point. These two packages combined allow robust segmentation of iPSCs solely on phase-contrast single-channel images and enable live imaging data to be easily integrated to endpoint data sets while retaining the dynamics of cellular responses. The described workflow facilitates characterization of the response of live-imaged iPSCs to external stimuli and definition of cell line-specific, phenotypic signatures. We present an efficient tool set for automated high-content analysis suitable for cells with challenging morphology. This approach has potentially widespread applications for human pluripotent stem cells and other cell types. © 2016 Society for Laboratory Automation and Screening.

  18. Analysis of receptor clustering on cell surfaces by imaging fluorescent particles.

    Science.gov (United States)

    Morrison, I E; Anderson, C M; Georgiou, G N; Stevenson, G V; Cherry, R J

    1994-09-01

    Fluorescently labeled low density lipoproteins (LDL) and influenza virus particles were bound to the surface of human fibroblasts and imaged with a cooled slow-scan CCD camera attached to a fluorescence microscope. Particles were also imaged after attachment to polylysine-coated microscope slides. The digital images were analyzed by fitting data points in the region of fluorescent spots by a two-dimensional Gaussian function, thus obtaining a measure of spot intensity with correction for local background. The intensity distributions for particles bound to polylysine slides were mainly accounted for by particle size distributions as determined by electron microscopy. In the case of LDL, the intensity distributions for particles bound to fibroblasts were considerably broadened, indicative of clustering. The on-cell intensity distributions were deconvolved into 1-particle, 2-particle, 3-particle, etc. components using the data obtained with LDL bound to polylysine-coated slides as an empirical measure of the single particle intensity distribution. This procedure yielded a reasonably accurate measure of the proportion of single particles, but large errors were encountered in the proportions of larger cluster sizes. The possibility of studying the dynamics of clustering was investigated by binding LDL to cells at 4 degrees C and observing changes in the intensity distribution with time after warming to 20 degrees C.

  19. Investigation of penetration force of living cell using an atomic force microscope

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Eun Young; Kim, Young Tae; Kim, Dae Eun [Yonsei University, Seoul (Korea, Republic of)

    2009-07-15

    Recently, the manipulation of a single cell has been receiving much attention in transgenesis, in-vitro fertilization, individual cell based diagnosis, and pharmaceutical applications. As these techniques require precise injection and manipulation of cells, issues related to penetration force arise. In this work the penetration force of living cell was studied using an atomic force microscope (AFM). L929, HeLa, 4T1, and TA3 HA II cells were used for the experiments. The results showed that the penetration force was in the range of 2{approx}22 nN. It was also found that location of cell penetration and stiffness of the AFM cantilever affected the penetration force significantly. Furthermore, double penetration events could be detected, due to the multi-membrane layers of the cell. The findings of this work are expected to aid in the development of precision micro-medical instruments for cell manipulation and treatment

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

    Energy Technology Data Exchange (ETDEWEB)

    Ewald, Dr. Maxime [University of Bourgogne, 21078 Dijon, France.; Tetard, Laurene [ORNL; Elie-Caille, Dr. Cecile [Institut FEMTO-ST UMR CNRS 6174, University Franche-Comté, 25044 Besancon, France; Nicod, Laurence [University of Franche-Comte, Laboratoire de Biologie Cellulaire; Passian, Ali [ORNL; Bourillot, Dr. Eric [University of Bourgogne, 21078 Dijon, France.; Lesniewska, Prof. Eric [University of Bourgogne, 21078 Dijon, France.

    2014-01-01

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

  1. A microfluidic platform for correlative live-cell and super-resolution microscopy.

    Directory of Open Access Journals (Sweden)

    Johnny Tam

    Full Text Available 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.

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

    Science.gov (United States)

    Padman, Benjamin S; Ramm, Georg

    2014-01-01

    Live-cell correlative light and electron microscopy permits the visualization of ultrastructure details associated with dynamic biological processes. On the optical level, fluorescence microscopy can be further combined with functional studies of intracellular processes and manipulation of biological samples using laser light. However, the major challenge is to relocate intracellular compartments in three dimensions after the sample has undergone an extensive EM sample preparation process. Here, we describe a detailed protocol for live-cell CLEM that provides easy guidance for 3D relocalization. Based on the use of the novel polymer film TOPAS as direct imaging substrate, we provide a setup that uses highly visible toner particles for tracking the region of interest in 2D and fiducial markers for the 3D relocation of intracellular structures. An example is given where a single mitochondria is targeted by laser microirradiation in live-cell fluorescence microscopy. After relocating the same structure in 3D in serial EM sections, the changes to the mitochondrial ultrastructure are observed by TEM. The method is suitable for correlation of live-cell microscopy of cells and can be performed using any inverted optical microscope. © 2014 Elsevier Inc. All rights reserved.

  3. Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles.

    Science.gov (United States)

    Seynhaeve, Ann L B; Ten Hagen, Timo L M

    2017-11-01

    Conventional 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 which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

  4. 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.

  5. Improved molecular toolkit for cAMP studies in live cells

    Directory of Open Access Journals (Sweden)

    Nicol Xavier

    2011-07-01

    Full Text Available Abstract Background cAMP is a ubiquitous second messenger involved in a wide spectrum of cellular processes including gene transcription, cell proliferation, and axonal pathfinding. Precise spatiotemporal manipulation and monitoring in live cells are crucial for investigation of cAMP-dependent pathways, but existing tools have several limitations. Findings We have improved the suitability of cAMP manipulating and monitoring tools for live cell imaging. We attached a red fluorescent tag to photoactivated adenylyl cyclase (PACα that enables reliable visualization of this optogenetic tool for cAMP manipulation in target cells independently of its photoactivation. We show that replacement of CFP/YFP FRET pair with GFP/mCherry in the Epac2-camps FRET probe reduces photobleaching and stabilizes the noise level during imaging experiments. Conclusions The modifications of PACα and Epac2-camps enhance these tools for in vitro cAMP studies in cultured living cells and in vivo studies in live animals in a wide range of experiments, and particularly for long term time-lapse imaging.

  6. Modular fluorescence complementation sensors for live cell detection of epigenetic signals at endogenous genomic sites.

    Science.gov (United States)

    Lungu, Cristiana; Pinter, Sabine; Broche, Julian; Rathert, Philipp; Jeltsch, Albert

    2017-09-21

    Investigation of the fundamental role of epigenetic processes requires methods for the locus-specific detection of epigenetic modifications in living cells. Here, we address this urgent demand by developing four modular fluorescence complementation-based epigenetic biosensors for live-cell microscopy applications. These tools combine engineered DNA-binding proteins with domains recognizing defined epigenetic marks, both fused to non-fluorescent fragments of a fluorescent protein. The presence of the epigenetic mark at the target DNA sequence leads to the reconstitution of a functional fluorophore. With this approach, we could for the first time directly detect DNA methylation and histone 3 lysine 9 trimethylation at endogenous genomic sites in live cells and follow dynamic changes in these marks upon drug treatment, induction of epigenetic enzymes and during the cell cycle. We anticipate that this versatile technology will improve our understanding of how specific epigenetic signatures are set, erased and maintained during embryonic development or disease onset.Tools for imaging epigenetic modifications can shed light on the regulation of epigenetic processes. Here, the authors present a fluorescence complementation approach for detection of DNA and histone methylation at endogenous genomic sites allowing following of dynamic changes of these marks by live-cell microscopy.

  7. Fourier transform infrared spectroscopy imaging of live epithelial cancer cells under non-aqueous media.

    Science.gov (United States)

    Soh, JunYi; Chueng, Adeline; Adio, Aminat; Cooper, Alan J; Birch, Brian R; Lwaleed, Bashir A

    2013-04-01

    Fourier transform infrared (FT-IR) imaging is increasingly being applied to biomedical specimens, but strong IR absorption by water complicates live cell imaging. This study investigates the viability of adherent epithelial cells maintained for short periods under mineral oils in order to facilitate live cell spectroscopy using FT-IR with subsequent imaging. The MGH-U1 urothelial or CaCo2 colorectal cancer cell lines were grown on plastic surfaces or mid-range infrared transparent windows. Medium in established cultures was replaced with paraffin mineral oil, or Fluorolube, for up to 2 h, and viability assessed by supravital staining. Drug handling characteristics were also assessed. Imaging of preparations was attempted by reflectance and transmission using a Varian FT-IR microscope. Cells covered by mineral oil remained viable for 2 h, with recovery into normal medium possible. MTT ((3-(4,5-dimethylthlazol-2-yl)-2,5-diphenyl tetrazolium) conversion to crystalline formazan and differential patterns of drug uptake were maintained. The combination of a calcium fluoride substrate, Fluorolube oil, and transmission optics proved best for spectroscopy. Spectral features were used to obtain images of live cells. The viability of cells overlaid with IR transparent oils was assessed as part of a technique to optimise conditions for FT-IR imaging. Images of untreated cells were obtained using both reflectance and transmission. This represents an effective means of imaging live cells by IR spectroscopy, and also means that imaging is not necessarily a terminal event. It also increases options for producing images based on real-time biochemistry in a range of in vitro experimental and 'optical biopsy' contexts.

  8. NKp46 clusters at the immune synapse and regulates NK cell polarization

    Directory of Open Access Journals (Sweden)

    Uzi eHadad

    2015-09-01

    Full Text Available Natural killer cells play an important role in first-line defense against tumor and virus-infected cells. The activity of NK cells is tightly regulated by a repertoire of cell-surface expressed inhibitory and activating receptors. NKp46 is a major NK cell activating receptor that is involved in the elimination of target cells. NK cells form different types of synapses that result in distinct functional outcomes: cytotoxic, inhibitory, and regulatory. Recent studies revealed that complex integration of NK receptor signaling controls cytoskeletal rearrangement and other immune synapse-related events. However the distinct nature by which NKp46 participates in NK immunological synapse formation and function remains unknown. In this study we determined that NKp46 forms microclusters structures at the immune synapse between NK cells and target cells. Over-expression of human NKp46 is correlated with increased accumulation of F-actin mesh at the immune synapse. Concordantly, knock-down of NKp46 in primary human NK cells decreased recruitment of F-actin to the synapse. Live cell imaging experiments showed a linear correlation between NKp46 expression and lytic granules polarization to the immune synapse. Taken together, our data suggest that NKp46 signaling directly regulates the NK lytic immune synapse from early formation to late function.

  9. Stem cell dynamics in muscle regeneration: Insights from live imaging in different animal models.

    Science.gov (United States)

    Ratnayake, Dhanushika; Currie, Peter D

    2017-06-01

    In recent years, live imaging has been adopted to study stem cells in their native environment at cellular resolution. In the skeletal muscle field, this has led to visualising the initial events of muscle repair in mouse, and the entire regenerative response in zebrafish. Here, we review recent discoveries in this field obtained from live imaging studies. Tracking of tissue resident stem cells, the satellite cells, following injury has captured the morphogenetic dynamics of stem/progenitor cells as they facilitate repair. Asymmetric satellite cell division generated a clonogenic progenitor pool, providing in vivo validation for this mechanism. Furthermore, there is an emerging role of stem/progenitor cell guidance at the injury site by cellular protrusions. This review concludes that live imaging is a critical tool for discovering the distinct processes that occur during regeneration, emphasising the importance of imaging in diverse animal models to capture the entire scope of stem cell functions. Also see the Video Abstract. Link to: https://youtube/tgUHSBD1N0g. © 2017 WILEY Periodicals, Inc.

  10. Fully synthetic polymer vesicles for intracellular delivery of antibodies in live cells.

    Science.gov (United States)

    Canton, Irene; Massignani, Marzia; Patikarnmonthon, Nisa; Chierico, Luca; Robertson, James; Renshaw, Stephen A; Warren, Nicholas J; Madsen, Jeppe P; Armes, Steven P; Lewis, Andrew L; Battaglia, Giuseppe

    2013-01-01

    There is an emerging need both in pharmacology and within the biomedical industry to develop new tools to target intracellular mechanisms. The efficient delivery of functionally active proteins within cells is potentially a powerful research strategy, especially through the use of antibodies. In this work, we report on a nanovector for the efficient encapsulation and delivery of antibodies into live cells with no significant loss of cell viability or any deleterious effect on cell metabolic activity. This delivery system is based on poly[2-(methacryloyloxy)ethyl phosphorylcholine]-block-[2-(diisopropylamino)ethyl methacrylate] (PMPC-PDPA), a pH-sensitive diblock copolymer that self-assembles to form nanometer-sized vesicles, also known as polymersomes, at physiological pH. Polymersomes can successfully deliver relatively high antibody payloads within different types of live cells. We demonstrate that these antibodies can target their respective epitope showing immunolabeling of γ-tubulin, actin, Golgi protein, and the transcription factor NF-κB in live cells. Finally, we demonstrate that intracellular delivery of antibodies can control specific subcellular events, as well as modulate cell activity and proinflammatory processes.

  11. 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.

  12. Live endothelial cells imaged by Scanning Near-field Optical Microscopy (SNOM): capabilities and challenges.

    Science.gov (United States)

    Bulat, Katarzyna; Rygula, Anna; Szafraniec, Ewelina; Ozaki, Yukihiro; Baranska, Malgorzata

    2017-06-01

    The scanning near-field optical microscopy (SNOM) shows a potential to study details of biological samples, since it provides the optical images of objects with nanometric spatial resolution (50-200 nm) and the topographic information at the same time. The goal of this work is to demonstrate the capabilities of SNOM in transmission configuration to study human endothelial cells and their morphological changes, sometimes very subtle, upon inflammation. Various sample preparations were tested for SNOM measurements and promising results are collected to show: 1) the influence of α tumor necrosis factor (TNF-α) on EA.hy 926 cells (measurements of the fixed cells); 2) high resolution images of various endothelial cell lines, i.e. EA.hy 926 and HLMVEC (investigations of the fixed cells in buffer environment); 3) imaging of live endothelial cells in physiological buffers. The study demonstrate complementarity of the SNOM measurements performed in air and in liquid environments, on fixed as well as on living cells. Furthermore, it is proved that the SNOM is a very useful method for analysis of cellular morphology and topography. Changes in the cell shape and nucleus size, which are the symptoms of inflammatory reaction, were noticed in TNF-α activated EA.hy 926 cells. The cellular structures of submicron size were observed in high resolution optical images of cells from EA.hy 926 and HLMVEC lines. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. A cluster randomised controlled trial of an occupational therapy intervention for residents with stroke living in UK care homes (OTCH: study protocol

    Directory of Open Access Journals (Sweden)

    Sackley Cath M

    2012-07-01

    Full Text Available Abstract Background The occupational therapy (OT in care homes study (OTCH aims to investigate the effect of a targeted course of individual OT (with task training, provision of adaptive equipment, minor environmental adaptations and staff education for stroke survivors living in care homes, compared to usual care. Methods/Design A cluster randomised controlled trial of United Kingdom (UK care homes (n = 90 with residents (n = 900 who have suffered a stroke or transient ischaemic attack (TIA, and who are not receiving end-of-life care. Homes will be stratified by centre and by type of care provided and randomised (50:50 using computer generated blocked randomisation within strata to receive either the OT intervention (3 months intervention from an occupational therapist or control (usual care. Staff training on facilitating independence and mobility and the use of adaptive equipment, will be delivered to every home, with control homes receiving this after the 12 month follow-up. Allocation will be concealed from the independent assessors, but the treating therapists, and residents will not be masked to the intervention. Measurements are taken at baseline prior to randomisation and at 3, 6 and 12 months post randomisation. The primary outcome measure is independence in self-care activities of daily living (Barthel Activities of Daily Living Index. Secondary outcome measures are mobility (Rivermead Mobility Index, mood (Geriatric Depression Scale, preference based quality of life measured from EQ-5D and costs associated with each intervention group. Quality adjusted life years (QALYs will be derived based on the EQ-5D scores. Cost effectiveness analysis will be estimated and measured by incremental cost effectiveness ratio. Adverse events will be recorded. Discussion This study will be the largest cluster randomised controlled trial of OT in care homes to date and will clarify the currently inconclusive literature on the efficacy of OT for

  14. Apathy and Its Response to Antipsychotic Review and Nonpharmacological Interventions in People With Dementia Living in Nursing Homes: WHELD, a Factorial Cluster Randomized Controlled Trial.

    Science.gov (United States)

    Rajkumar, Anto P; Ballard, Clive; Fossey, Jane; Corbett, Anne; Woods, Bob; Orrell, Martin; Prakash, Rohan; Moniz-Cook, Esme; Testad, Ingelin

    2016-08-01

    Apathy is common, impactful, and difficult to manage in people with dementia. We evaluated the efficacy of nonpharmacological interventions, exercise, and social interaction, in combination with antipsychotic review, to reduce apathy in people with dementia living in nursing homes in a cluster randomized controlled trial (RCT). Well-being and health for people with dementia (WHELD) program included a 2 × 2 × 2 factorial cluster RCT involving people with dementia living in 16 nursing homes in the United Kingdom. All homes received training in person-centered care, and were randomized to receive antipsychotic review, social interaction, and exercise, either alone or in combinations. Apathy was one of the secondary outcomes of the WHELD trial, and it was measured by the Neuropsychiatric Inventory-nursing home version at baseline and 9 months (n = 273). We used multilevel mixed effects linear regression models to assess the impact of the interventions on apathy. Prevalence of apathy was 44.0% (n = 120; 95% confidence interval [CI] 38.1%-49.9%) at baseline. Severity of apathy had significant positive correlations with dementia severity, neuropsychiatric symptoms, depressive symptoms, agitation, and the needs of the people with dementia (P apathy (β = 5.37; SE = 0.91; P apathy. Antipsychotic review can play a significant role in improving apathy in people with dementia living in nursing homes, when combined with psychosocial interventions such as social interaction and exercise. Guidance must be adapted to reflect this subtlety in care. Copyright © 2016 AMDA – The Society for Post-Acute and Long-Term Care Medicine. Published by Elsevier Inc. All rights reserved.

  15. The chronically inflamed central nervous system provides niches for long-lived plasma cells.

    Science.gov (United States)

    Pollok, Karolin; Mothes, Ronja; Ulbricht, Carolin; Liebheit, Alina; Gerken, Jan David; Uhlmann, Sylvia; Paul, Friedemann; Niesner, Raluca; Radbruch, Helena; Hauser, Anja Erika

    2017-11-25

    Although oligoclonal bands in the cerebrospinal fluid have been a hallmark of multiple sclerosis diagnosis for over three decades, the role of antibody-secreting cells in multiple sclerosis remains unclear. T and B cells are critical for multiple sclerosis pathogenesis, but increasing evidence suggests that plasma cells also contribute, through secretion of autoantibodies. Long-lived plasma cells are known to drive various chronic inflammatory conditions as e.g. systemic lupus erythematosus, however, to what extent they are present in autoimmune central nervous system inflammation has not yet been investigated. In brain biopsies from multiple sclerosis patients and other neurological diseases, we could detect non-proliferating plasma cells (CD138 + Ki67 - ) in the parenchyma. Based on this finding, we hypothesized that long-lived plasma cells can persist in the central nervous system (CNS). In order to test this hypothesis, we adapted the multiple sclerosis mouse model experimental autoimmune encephalomyelitis to generate a B cell memory response. Plasma cells were found in the meninges and the parenchyma of the inflamed spinal cord, surrounded by tissue areas resembling survival niches for these cells, characterized by an up-regulation of chemokines (CXCL12), adhesion molecules (VCAM-1) and survival factors (APRIL and BAFF). In order to determine the lifetime of plasma cells in the chronically inflamed CNS, we labeled the DNA of proliferating cells with 5-ethynyl-2'-deoxyuridine (EdU). Up to five weeks later, we could detect EdU + long-lived plasma cells in the murine CNS. To our knowledge, this is the first study describing non-proliferating plasma cells directly in the target tissue of a chronic inflammation in humans, as well as the first evidence demonstrating the ability of plasma cells to persist in the CNS, and the ability of the chronically inflamed CNS tissue to promote this persistence. Hence, our results suggest that the CNS provides survival niches for

  16. 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.

  17. Dancoff factors of unit cells in cluster geometry with partial absorption of neutrons

    International Nuclear Information System (INIS)

    Rodrigues, Leticia Jenisch

    2011-01-01

    In its classical formulation, the Dancoff factor for a perfectly absorbing fuel rod is defined as the relative reduction in the incurrent of resonance neutrons into the rod in the presence of neighboring rods, as compared to the incurrent into a single fuel rod immersed in an infinite moderator. Alternatively, this factor can be viewed as the probability that a neutron emerging from the surface of a fuel rod will enter another fuel rod without any collision in the moderator or cladding. For perfectly absorbing fuel these definitions are equivalent. In the last years, several works appeared in literature reporting improvements in the calculation of Dancoff factors, using both the classical and the collision probability definitions. In this work, we step further reporting Dancoff factors for perfectly absorbing (Black) and partially absorbing (Grey) fuel rods calculated by the collision probability method, in cluster cells with square outer boundaries. In order to validate the results, comparisons are made with the equivalent cylindricalized cell in hypothetical test cases. The calculation is performed considering specularly reflecting boundary conditions, for the square lattice, and diffusive reflecting boundary conditions, for the cylindrical geometry. The results show the expected asymptotic behavior of the solution with increasing cell sizes. In addition, Dancoff factors are computed for the Canadian cells CANDU-37 and CANFLEX by the Monte Carlo and Direct methods. Finally, the effective multiplication factors, k eff , for these cells (cluster cell with square outer boundaries and the equivalent cylindricalized cell) are also computed, and the differences reported for the cases using the perfect and partial absorption assumptions. (author)

  18. Functional dissection of HOXD cluster genes in regulation of neuroblastoma cell proliferation and differentiation.

    Directory of Open Access Journals (Sweden)

    Yunhong Zha

    Full Text Available Retinoic acid (RA can induce growth arrest and neuronal differentiation of neuroblastoma cells and has been used in clinic for treatment of neuroblastoma. It has been reported that RA induces the expression of several HOXD genes in human neuroblastoma cell lines, but their roles in RA action are largely unknown. The HOXD cluster contains nine genes (HOXD1, HOXD3, HOXD4, and HOXD8-13 that are positioned sequentially from 3' to 5', with HOXD1 at the 3' end and HOXD13 the 5' end. Here we show that all HOXD genes are induced by RA in the human neuroblastoma BE(2-C cells, with the genes located at the 3' end being activated generally earlier than those positioned more 5' within the cluster. Individual induction of HOXD8, HOXD9, HOXD10 or HOXD12 is sufficient to induce both growth arrest and neuronal differentiation, which is associated with downregulation of cell cycle-promoting genes and upregulation of neuronal differentiation genes. However, induction of other HOXD genes either has no effect (HOXD1 or has partial effects (HOXD3, HOXD4, HOXD11 and HOXD13 on BE(2-C cell proliferation or differentiation. We further show that knockdown of HOXD8 expression, but not that of HOXD9 expression, significantly inhibits the differentiation-inducing activity of RA. HOXD8 directly activates the transcription of HOXC9, a key effector of RA action in neuroblastoma cells. These findings highlight the distinct functions of HOXD genes in RA induction of neuroblastoma cell differentiation.

  19. A fluorogenic probe for imaging protein S-nitrosylation in live cells.

    Science.gov (United States)

    Shao, Shiyi; Chen, Bo; Cheng, Juan; Wang, Chengkun; Zhang, Yanli; Shao, Lingxiao; Hu, Yongzhou; Han, Yifeng; Han, Feng; Li, Xin

    2017-08-15

    S-nitrosylation is a posttranslational modification of protein cysteine residues leading to the formation of S-nitrosothiols and its detection is crucial to understanding of redox regulation and NO-based signaling. Prototypical detection methods for S-nitrosylation are always carried out ex situ. However, the reversible nature and the tendency of transnitrosylation highlight the necessity of its probing in intact live biological contexts. Herein we provide a fluorogenic chemical probe for the detection of S-nitrosylation in live endothelial cells. The probe is weakly emissive alone and becomes highly fluorescent only after undergoing a reaction with S-nitrosothiols in live cellular environments. This probe features high degrees of specificity and desirable sensitivity. Furthermore, it has been successfully applied to image the dynamic change of protein S-nitrosylation in live endothelial cells. The applicability of the probe in complex biological systems has been additionally verified by imaging a known target of S-nitrosylation, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in live cells. Due to the versatility exemplified, this probe holds great promise for exploring the role of protein S-nitrosylation in the pathophysiological process of a variety of vascular diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Live imaging of innate immune cell sensing of transformed cells in zebrafish larvae: parallels between tumor initiation and wound inflammation.

    Directory of Open Access Journals (Sweden)

    Yi Feng

    Full Text Available It has not previously been possible to live image the earliest interactions between the host environment and oncogene-transformed cells as they initiate formation of cancers within an organism. Here we take advantage of the translucency of zebrafish larvae to observe the host innate immune cell response as oncogene-transformed melanoblasts and goblet cells multiply within the larval skin. Our studies indicate activation of leukocytes at very early stages in larvae carrying a transformed cell burden. Locally, we see recruitment of neutrophils and macrophages by 48 h post-fertilization, when transformed cells are still only singletons or doublets, and soon after this we see intimate associations between immune and transformed cells and frequent examples of cytoplasmic tethers linking the two cell types, as well as engulfment of transformed cells by both neutrophils and macrophages. We show that a major component of the signal drawing inflammatory cells to oncogenic HRAS(G12V-transformed cells is H(2O(2, which is also a key damage cue responsible for recruiting neutrophils to a wound. Our short-term blocking experiments show that preventing recruitment of immune cells at these early stages results in reduced growth of transformed cell clones and suggests that immune cells may provide a source of trophic support to the transformed cells just as they do at a site of tissue repair. These parallels between the inflammatory responses to transformed cells and to wounds reinforce the suggestion by others that cancers resemble non-healing wounds.

  1. 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

  2. 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.

  3. 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.

  4. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators....

  5. Synthesis and functionalization of nano- and micro-particles for sensing and therapy in living cells

    OpenAIRE

    Penon Esteva, Oriol

    2013-01-01

    In the present thesis Supramolecular chemistry is exploited to approach applications in the area of Nanomedicine, and it is, focused on the design and preparation of different micro and nanotools for sensing and therapy, in living cells. Initially, the combination of silicon surface chemistry with the incorporation of bioactive molecules has been investigated in order to obtain a potentially microtool suitable for cell tagging. Furthermore, the design and synthesis of organic compounds as int...

  6. 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

  7. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    Science.gov (United States)

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  8. 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.

  9. Vinylboronic Acids as Efficient Bioorthogonal Reactants for Tetrazine Labeling in Living Cells.

    Science.gov (United States)

    Eising, Selma; van der Linden, Nicole G A; Kleinpenning, Fleur; Bonger, Kimberly M

    2018-02-19

    Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality present in the cell. The tetrazine ligation is very suitable as a bioorthogonal reaction because of its selectivity and high reaction rates with several alkenes and alkynes. Recently, we described vinylboronic acids (VBAs) as novel hydrophilic bioorthogonal moieties that react efficiently with dipyridyl-s-tetrazines and used them for protein modification in cell lysate. It is not clear, however, whether VBAs are suitable for labeling experiments in living cells because of the possible coordination with, for example, vicinal carbohydrate diols. Here, we evaluated VBAs as bioorthogonal reactants for labeling of proteins in living cells using an irreversible inhibitor of the proteasome and compared the reactivity to that of an inhibitor containing norbornene, a widely used reactant for the tetrazine ligation. No large differences were observed between the VBA and norbornene probes in a two-step labeling approach with a cell-penetrable fluorescent tetrazine, indicating that the VBA gives little or no side reactions with diols and can be used efficiently for protein labeling in living cells.

  10. 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.

  11. Microenvironments and different nanoparticle dynamics in living cells revealed by a standard nanoparticle.

    Science.gov (United States)

    Pack, Chan Gi; Song, Mi Ryoung; Tae, Eunju Lee; Hiroshima, Michio; Byun, Kyung Hee; Kim, Jun Sung; Sako, Yasushi

    2012-11-10

    For quantitative analysis of nanoparticle diffusions and submicro-environments in living cells, use of newly synthesized silica-based fluorescent nanoparticle (Si-FNP) as a standard nanoprobe is successfully demonstrated. The appropriate characteristics of a standard probe were fully analyzed in vitro by single molecule detection, transmission electron microscopy, and dynamic light scattering. Using fluorescence correlation analysis in single living cells, we quantitatively compared the diffusional properties of the standard Si-FNP with a diameter of 50 nm, peptide coated Si-FNP, streptavidin coated Qdot, and GFP molecule which have different sizes and surface properties. The result demonstrates that the standard Si-FNP without coat is minimally trapped in the vesicles in the process of cellular endocytosis. Interestingly, a large proportion of Si-FNP introduced into the cells by electroporation diffuses freely in the cells during a cell cycle suggesting free diffusing NPs are hardly trapped in the vesicles. The simple but highly sensitive method will provide insight into strategies to understanding the hydrodynamic process of nanoparticle delivery into living cells as well as the cellular microenvironment in the view of submicro-size. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Bhanu Neupane

    2015-09-01

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

  13. Rapid telomere motions in live human cells analyzed by highly time-resolved microscopy

    Directory of Open Access Journals (Sweden)

    Wang Xueying

    2008-10-01

    Full Text Available Abstract Background Telomeres cap chromosome ends and protect the genome. We studied individual telomeres in live human cancer cells. In capturing telomere motions using quantitative imaging to acquire complete high-resolution three-dimensional datasets every second for 200 seconds, telomere dynamics were systematically analyzed. Results The motility of individual telomeres within the same cancer cell nucleus was widely heterogeneous. One class of internal heterochromatic regions of chromosomes analyzed moved more uniformly and showed less motion and heterogeneity than telomeres. The single telomere analyses in cancer cells revealed that shorter telomeres showed more motion, and the more rapid telomere motions were energy dependent. Experimentally increasing bulk telomere length dampened telomere motion. In contrast, telomere uncapping, but not a DNA damaging agent, methyl methanesulfonate, significantly increased telomere motion. Conclusion New methods for seconds-scale, four-dimensional, live cell microscopic imaging and data analysis, allowing systematic tracking of individual telomeres in live cells, have defined a previously undescribed form of telomere behavior in human cells, in which the degree of telomere motion was dependent upon telomere length and functionality.

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

    Science.gov (United States)

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

    2016-08-01

    Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging. © 2016 The Authors.

  15. 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

  16. High Spatial Resolution Imaging of Endogenous Hydrogen Peroxide in Living Cells by Solid-State Fluorescence.

    Science.gov (United States)

    Lindberg, Eric; Winssinger, Nicolas

    2016-09-02

    Herein, we describe selective imaging of hydrogen peroxide using a precipitating dye conjugated to a boronic acid-based immolative linker. We achieved visualization of endogenous hydrogen peroxide in phagosomes by solid-state two-photon fluorescence imaging in living cells with exceptionally high spatial resolution. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Heterotrophic free-living and particle-bound bacterial cell size in the ...

    Indian Academy of Sciences (India)

    Regression analysis revealed that 18% of the variation in mean heterotrophic free-living bacterial cell size was due to biological oxygen demand (BOD) in the river Arkavathy, 11% due to surface water velocity (SWV) in the river Cauvery and 11% due to temperature in the river Kapila. Heterotrophic particle-bound bacterial ...

  18. Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell

    OpenAIRE

    Hein, Birka; Willig, Katrin I.; Hell, Stefan W.

    2008-01-01

    We demonstrate far-field optical imaging with subdiffraction resolution of the endoplasmic reticulum (ER) in the interior of a living mammalian cell. The diffraction barrier is overcome by applying stimulated emission depletion (STED) on a yellow fluorescent protein tag. Imaging individual structural elements of the ER revealed a focal plane (x, y) resolution of

  19. Tracking the Dynamic Folding and Unfolding of RNA G-Quadruplexes in Live Cells.

    Science.gov (United States)

    Chen, Xiu-Cai; Chen, Shuo-Bin; Dai, Jing; Yuan, Jia-Hao; Ou, Tian-Miao; Huang, Zhi-Shu; Tan, Jia-Heng

    2018-04-16

    Because of the absence of methods for tracking RNA G-quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G-quadruplexes is so far limited. Herein, we report a new red-emitting fluorescent probe, QUMA-1, for the selective, continuous, and real-time visualization of RNA G-quadruplexes in live cells. The applications of QUMA-1 in several previously intractable applications, including live-cell imaging of the dynamic folding, unfolding, and movement of RNA G-quadruplexes and the visualization of the unwinding of RNA G-quadruplexes by RNA helicase have been demonstrated. Notably, our real-time results revealed the complexity of the dynamics of RNA G-quadruplexes in live cells. We anticipate that the further application of QUMA-1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G-quadruplexes will uncover more information about the biological roles of RNA G-quadruplexes. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Nutritional status, quality of life and CD4 cell count of adults living ...

    African Journals Online (AJOL)

    Original Research: Nutritional status, quality of life and CD4 cell count of adults living with HIV/AIDS. 2009;22(3). S Afr J Clin Nutr a Venter E, b Gericke GJ, c Bekker PJ a Division of Human Nutrition, Faculty of Health Sciences, Stellenbosch University b Department of Human Nutrition, Faculty of Health Sciences, University ...

  1. Photocaged Nanoparticle Sensor for Sensitive MicroRNA Imaging in Living Cancer Cells with Temporal Control.

    Science.gov (United States)

    Shen, Yi; Li, Zhi; Wang, Ganglin; Ma, Nan

    2018-02-23

    Sensitive imaging of microRNA in living cells is of great value for disease diagnostics and prognostics. While signal amplification-based strategies have been developed for imaging low-abundance disease-relevant microRNA molecules, precise temporal control over sensor activity in living cells still remains a challenge, and limits their applications for sensing microRNA concentration dynamics. Herein, we report a class of photocaged nanoparticle sensors for highly sensitive imaging of microRNA in living cells with temporal control. The sensor features a DNA-templated gold nanoparticle-quantum dot satellite nanostructure which is temporarily inactivated by a photocaged DNA mask. Upon UV light irradiation, the sensor restores its activity for catalytic sensing of microRNA in living cells via entropy-driven two-step toehold-mediated strand displacement reactions. We show that the sensor exhibits quick response to UV light, robust intracellular stability, and high specificity and sensitivity for the microRNA target. On the basis of this strategy, precise control over sensor activity is achieved using an external light trigger, where on-demand sensing could be potentially performed with spatiotemporal control.

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

    Science.gov (United States)

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

    2014-07-01

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

  3. Synthesis and live-cell imaging of fluorescent sterols for analysis of intracellular cholesterol transport

    DEFF Research Database (Denmark)

    Modzel, Maciej; Lund, Frederik W.; Wüstner, Daniel

    2017-01-01

    Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol...

  4. 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

  5. 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

  6. Live-cell imaging to measure BAX recruitment kinetics to mitochondria during apoptosis

    Science.gov (United States)

    Maes, Margaret E.; Schlamp, Cassandra L.

    2017-01-01

    The pro-apoptotic BCL2 gene family member, BAX, plays a pivotal role in the intrinsic apoptotic pathway. Under cellular stress, BAX recruitment to the mitochondria occurs when activated BAX forms dimers, then oligomers, to initiate mitochondria outer membrane permeabilization (MOMP), a process critical for apoptotic progression. The activation and recruitment of BAX to form oligomers has been studied for two decades using fusion proteins with a fluorescent reporter attached in-frame to the BAX N-terminus. We applied high-speed live cell imaging to monitor the recruitment of BAX fusion proteins in dying cells. Data from time-lapse imaging was validated against the activity of endogenous BAX in cells, and analyzed using sigmoid mathematical functions to obtain detail of the kinetic parameters of the recruitment process at individual mitochondrial foci. BAX fusion proteins behave like endogenous BAX during apoptosis. Kinetic studies show that fusion protein recruitment is also minimally affected in cells lacking endogenous BAK or BAX genes, but that the kinetics are moderately, but significantly, different with different fluorescent tags in the fusion constructs. In experiments testing BAX recruitment in 3 different cell lines, our results show that regardless of cell type, once activated, BAX recruitment initiates simultaneously within a cell, but exhibits varying rates of recruitment at individual mitochondrial foci. Very early during BAX recruitment, pro-apoptotic molecules are released in the process of MOMP, but different molecules are released at different times and rates relative to the time of BAX recruitment initiation. These results provide a method for BAX kinetic analysis in living cells and yield greater detail of multiple characteristics of BAX-induced MOMP in living cells that were initially observed in cell free studies. PMID:28880942

  7. 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.

  8. 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

  9. 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.

  10. Cell-Penetrating Ability of Peptide Hormones: Key Role of Glycosaminoglycans Clustering

    Directory of Open Access Journals (Sweden)

    Armelle Tchoumi Neree

    2015-11-01

    Full Text Available Over the last two decades, the potential usage of cell-penetrating peptides (CPPs for the intracellular delivery of various molecules has prompted the identification of novel peptidic identities. However, cytotoxic effects and unpredicted immunological responses have often limited the use of various CPP sequences in the clinic. To overcome these issues, the usage of endogenous peptides appears as an appropriate alternative approach. The hormone pituitary adenylate-cyclase-activating polypeptide (PACAP38 has been recently identified as a novel and very efficient CPP. This 38-residue polycationic peptide is a member of the secretin/glucagon/growth hormone-releasing hormone (GHRH superfamily, with which PACAP38 shares high structural and conformational homologies. In this study, we evaluated the cell-penetrating ability of cationic peptide hormones in the context of the expression of cell surface glycosaminoglycans (GAGs. Our results indicated that among all peptides evaluated, PACAP38 was unique for its potent efficiency of cellular uptake. Interestingly, the abilities of the peptides to reach the intracellular space did not correlate with their binding affinities to sulfated GAGs, but rather to their capacity to clustered heparin in vitro. This study demonstrates that the uptake efficiency of a given cationic CPP does not necessarily correlate with its affinity to sulfated GAGs and that its ability to cluster GAGs should be considered for the identification of novel peptidic sequences with potent cellular penetrating properties.

  11. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...... of a large panel of other tissue and cell types, and transcripts with beta cell-abundant and -selective expression were identified. Iteration of this analysis in mouse, rat and human tissues generated a panel of conserved beta cell biomarkers. This panel was then used to compare isolated versus laser capture...... microdissected beta cells, monitor adaptations of the beta cell phenotype to fasting, and retrieve possible conserved transcriptional regulators....

  12. Geometric effect of the hydrogel grid structure on in vitro formation of homogeneous MIN6 cell clusters.

    Science.gov (United States)

    Bae, Chae Yun; Min, Mun-kyeong; Kim, Hail; Park, Je-Kyun

    2014-07-07

    A microstructure-based hydrogel was employed to study the relationship between spatial specificity and cellular behavior, including cell fate, proliferation, morphology, and insulin secretion in pancreatic β-cells. To effectively form homogeneous cell clusters in vitro, we made cell-containing hydrogel membrane constructs with an adapted grid structure based on a hexagonal micropattern. Homogeneous cell clusters (average diameter: 83.6 ± 14.2 μm) of pancreatic insulinoma (MIN6) cells were spontaneously generated in the floating hydrogel membrane constructs, including a hexagonal grid structure (size of cavity: 100 μm, interval between cavities: 30 μm). Interestingly, 3D clustering of MIN6 cells mimicking the structure of pancreatic islets was coalesced into a merged aggregate attaching to each hexagonal cavity of the hydrogel grid structure. The fate and insulin secretion of homogeneous cell clusters in the hydrogel grid structure were also assessed. The results of these designable hydrogel-cell membrane constructs suggest that facultative in vitro β-cell proliferation and maintenance can be applied to biofunctional assessments.

  13. Innovative regions and industrial clusters in hydrogen and fuel cell technology

    DEFF Research Database (Denmark)

    Madsen, Anne Nygaard; Andersen, Per Dannemand

    2010-01-01

    Regional governments in Europe seem to be playing an increasing role in hydrogen and fuel cell (H2FC) development. A number of regions are supporting demonstration projects and building networks among regional stakeholders to strengthen their engagement in H2FC technology. In this article, we...... will analyse regions that are highly engaged in H2FC activity, based on three indicators: existing hydrogen infrastructure and production sites, general innovativeness and the presence of industrial clusters with relevance for H2FC. Our finding is that regions with high activity in H2FC development are also...

  14. Clusters of conserved beta cell marker genes for assessment of beta cell phenotype

    DEFF Research Database (Denmark)

    Martens, Geert A; Jiang, Lei; Hellemans, Karine H

    2011-01-01

    The aim of this study was to establish a gene expression blueprint of pancreatic beta cells conserved from rodents to humans and to evaluate its applicability to assess shifts in the beta cell differentiated state. Genome-wide mRNA expression profiles of isolated beta cells were compared to those...

  15. Diversity oriented fluorescence library approach (DOFLA) for live cell imaging probe development.

    Science.gov (United States)

    Yun, Seong-Wook; Kang, Nam-Young; Park, Sung-Jin; Ha, Hyung-Ho; Kim, Yun Kyung; Lee, Jun-Seok; Chang, Young-Tae

    2014-04-15

    A cell is the smallest functional unit of life. All forms of life rely on cellular processes to maintain normal functions, and changes in cell function induced by metabolic disturbances, physicochemical damage, infection, or abnormal gene expression may cause disease. To understand basic biology and to develop therapeutics for diseases, researchers need to study live cells. Along with advances in fluorescence microscopy and in vitro cell culture, live-cell imaging has become an essential tool in modern biology for the study of molecular and cellular events. Although researchers have often used fluorescent proteins to visualize cell-type-specific markers, this method requires genetic manipulations, which may not be appropriate in nontransgenic cells. Immunodetection of cellular markers requires the use of xenogenic antibodies, which may not detect intracellular markers in live cells. One option for overcoming these problems is the use of fluorescent small molecules targeted to specific cell types, which can enter live cells and interact with molecules of interest. We have used combinatorial chemistry to develop a large number of fluorescent small molecules as new imaging probes even without prior information about the probes' binding targets and mechanism, a strategy that we call the diversity oriented fluorescence library approach (DOFLA). We have used DOFLA to produce novel sensors and probes that detect a variety of biological and chemical molecules in vivo as well as in vitro. In this Account, we describe a series of fluorescent small molecules developed using DOFLA that bind specifically to particular cell types. These molecules provide new ways to detect and isolate these cells. The fluorescent probes CDy1, CDg4, and CDb8 tag embryonic stem cells and induced pluripotent stem cells but not fibroblasts or germ-line cells. CDr3 binds to an intracellular neural stem cell marker, fatty acid binding protein 7, which allows researchers to separate neural stem cells

  16. 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.)

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

    Directory of Open Access Journals (Sweden)

    Ken-Ichi Wada

    2017-12-01

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

  19. 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.

  20. 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 .

  1. 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}.

  2. 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.

  3. 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

  4. TRAIL death receptor 4 signaling via lysosome fusion and membrane raft clustering in coronary arterial endothelial cells: evidence from ASM knockout mice.

    Science.gov (United States)

    Li, Xiang; Han, Wei-Qing; Boini, Krishna M; Xia, Min; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4 (DR4), have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MR) clustering and the formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and co-localized with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1 (+/+)) mice. Furthermore, TRAIL triggered ASM translocation, ceramide production, and NADPH oxidase aggregation in MR clusters in Smpd1 ( +/+ ) CAECs, whereas these observations were not found in Smpd1 (-/-) CAECs. Moreover, ASM deficiency reduced TRAIL-induced O(2) (-[Symbol: see text]) production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer, we found that Lamp-1 (lysosome membrane marker protein) and ganglioside G(M1) (MR marker) were trafficking together in Smpd1 (+/+) CAECs, which was absent in Smpd1 (-/-) CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1 (-/-) CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking, membrane fusion and formation of MR redox signaling platforms

  5. Monitoring programmed cell death of living plant tissues in microfluidics using electrochemical and optical techniques

    DEFF Research Database (Denmark)

    Mark, Christina; Heiskanen, Arto; Svensson, Birte

    or optically detectable events during PCD in barley aleurone layer, a cell model for living plant tissues, for a better understanding of the underlying mechanisms of PCD in plants. Microfluidic cell culture enables in vitro experiments to approach in vivo conditions. The major advantage of electrochemical......Programmed cell death (PCD) in plants can influence the outcome of yield and quality of crops through its important role in seed germination and the defence process against pathogens. The main scope of the project is to apply microfluidic cell culture for the measurement of electrochemically......, since it is known that reactive oxygen species, which are affected by changes in the redox activity of the cells3, are involved in PCD in plants, but the relationship between and mechanisms behind ROS and PCD is only poorly understood in plant cells4. Recently, it has been shown, using optical detection...

  6. 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

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

    Science.gov (United States)

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

    2016-04-04

    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.

  8. 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.

  9. Picosecond fluorescence lifetime imaging microscope for imaging of living glioma cells

    Science.gov (United States)

    Fang, Qiyin; Wang, Jingjing; Sun, Yinghua; Vernier, Thomas; Papaioannou, Thanassis; Jo, Javier; Thu, Mya M.; Gundersen, Martin A.; Marcu, Laura

    2005-03-01

    In this communication, we report the imaging of living glioma cells using fluorescence lifetime imaging (FLIM) technique. The growing interests in developing novel techniques for diagnosis and minimally invasive therapy of brain tumor have led to microscopic studies of subcellular structures and intracellular processes in glioma cells. Fluorescence microscopy has been used with a number of exogenous molecular probes specific for certain intracellular structures such as mitochondria, peripheral benzodiazepine receptor (PBR), and calcium concentration. When probes with overlapping emission spectra being used, separate samples are required to image each probe individually under conventional fluorescence microscopy. We have developed a wide-field FLIM microscope that uses fluorescence lifetime as an additional contrast for resolving multiple markers in the same essay. The FLIM microscope consists of a violet diode laser and a nitrogen-pumped dye laser to provide tunable sub-nanosecond excitation from UV to NIR. The detection system is based on a time-gated ICCD camera with minimum 80 ps gate width. The performance of the system was evaluated using fluorescence dyes with reported lifetime values. Living rat glioma C6 cells were stained with JC-1 and Rhodamine 123. FLIM images were acquired and their lifetimes in living cells were found in good agreements with values measured in solutions by a time-domain fluorescence spectrometer. These results indicate that imaging of glioma cells using FLIM can resolve multiple spectrally-overlapping probes and provide quantitative functional information about the intracellular environment.

  10. A fluorescent carbon-dots-based mitochondria-targetable nanoprobe for peroxynitrite sensing in living cells.

    Science.gov (United States)

    Wu, Xiaoxue; Sun, Shan; Wang, Yuhui; Zhu, Jiali; Jiang, Kai; Leng, Yumin; Shu, Qinghai; Lin, Hengwei

    2017-04-15

    Mitochondria, the power generators in cell, are a primary organelle of oxygen consumption and a main source of reactive oxygen/nitrogen species (ROS/RNS). Peroxynitrite (ONOO - ), known as a kind of RNS, has been considered to be a significant factor in many cell-related biological processes, and there is great desire to develop fluorescent probes that can sensitively and selectively detect peroxynitrite in living cells. Herein, we developed a fluorescent carbon-dots (C-dots) based mitochondria-targetable nanoprobe with high sensitivity and selectivity for peroxynitrite sensing in living cells. The C-dots with its surface rich in amino groups was synthesized using o-phenylenediamine as carbon precursor, and it could be covalently conjugated with a mitochondria-targeting moiety, i.e. triphenylphosphonium (TPP). In the presence of peroxynitrite, the fluorescence of the constructed nanoprobe (C-dots-TPP) was efficiently quenched via a mechanism of photoinduced electron transfer (PET). The nanoprobe exhibited relatively high sensitivity (limit of detection: 13.5nM) and selectivity towards peroxynitrite in aqueous buffer. The performance of the nanoprobe for fluorescence imaging of peroxynitrite in mitochondria was investigated. The results demonstrated that the nanoprobe showed fine mitochondria-targeting ability and imaging contrast towards peroxynitrite in living cells. We anticipate that the proposed nanoprobe will provide a facile tool to explore the role played by peroxynitrite in cytobiology. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Scaffolds fabricated by 3D two-photon photopolymerization for live cell studies

    Science.gov (United States)

    Teplicky, T.; Cunderlikova, B.; Mateasik, A.; Vincze, A.; Chorvat, D.; Marcek Chorvatova, A.

    2016-12-01

    Design and fabrication of appropriate biocompatible microstructures that ensure fixation and control of experimental conditions for live cell and bacteria observations is an important prerequisite for number of real time experiments. Our approach is to design engineered microfabricated 3D structures for growth of cells in culture without significant modification of their metabolic state. Presented approach is aimed at evaluation of the potential applicability of biocompatible constructs in the biomedical field and thus live cell monitoring in controlled conditions. Design and evaluation of properties of materials and structures with mesoscopic arrangement and their interaction with biological objects is a prerequisite for establishment of physiologically relevant in vitro models of pathologies as well as for development of a new generation of nano / micro / bio-sensors.

  12. 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.

  13. Subcellular Localization of Thiol-Capped CdTe Quantum Dots in Living Cells

    Directory of Open Access Journals (Sweden)

    Chen Ji-Yao

    2009-01-01

    Full Text Available Abstract Internalization and dynamic subcellular distribution of thiol-capped CdTe quantum dots (QDs in living cells were studied by means of laser scanning confocal microscopy. These unfunctionalized QDs were well internalized into human hepatocellular carcinoma and rat basophilic leukemia cells in vitro. Co-localizations of QDs with lysosomes and Golgi complexes were observed, indicating that in addition to the well-known endosome-lysosome endocytosis pathway, the Golgi complex is also a main destination of the endocytosed QDs. The movement of the endocytosed QDs toward the Golgi complex in the perinuclear region of the cell was demonstrated.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging.

    Science.gov (United States)

    Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

    2017-10-15

    Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging. © 2017 Schvartz et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  20. A cluster-randomised trial of staff education to improve the quality of life of people with dementia living in residential care: the DIRECT study.

    Science.gov (United States)

    Beer, Christopher; Horner, Barbara; Flicker, Leon; Scherer, Samuel; Lautenschlager, Nicola T; Bretland, Nick; Flett, Penelope; Schaper, Frank; Almeida, Osvaldo P

    2011-01-01

    The Dementia In Residential care: EduCation intervention Trial (DIRECT) was conducted to determine if delivery of education designed to meet the perceived need of GPs and care staff improves the quality of life of participants with dementia living in residential care. This cluster-randomised controlled trial was conducted in 39 residential aged care facilities in the metropolitan area of Perth, Western Australia. 351 care facility residents aged 65 years and older with Mini-Mental State Examination ≤ 24, their GPs and facility staff participated. Flexible education designed to meet the perceived needs of learners was delivered to GPs and care facility staff in intervention groups. The primary outcome of the study was self-rated quality of life of participants with dementia, measured using the QOL-Alzheimer's Disease Scale (QOL-AD) at 4 weeks and 6 months after the conclusion of the intervention. Analysis accounted for the effect of clustering by using multi-level regression analysis. Education of GPs or care facility staff did not affect the primary outcome at either 4 weeks or 6 months. In a post hoc analysis excluding facilities in which fewer than 50% of staff attended an education session, self-rated QOL-AD scores were 6.14 points (adjusted 95%CI 1.14, 11.15) higher at four-week follow-up among residents in facilities randomly assigned to the education intervention. The education intervention directed at care facilities or GPs did not improve the quality of life ratings of participants with dementia as a group. This may be explained by the poor adherence to the intervention programme, as participants with dementia living in facilities where staff participated at least minimally seemed to benefit. ANZCTR.org.au ACTRN12607000417482.

  1. A cluster-randomised trial of staff education to improve the quality of life of people with dementia living in residential care: the DIRECT study.

    Directory of Open Access Journals (Sweden)

    Christopher Beer

    Full Text Available BACKGROUND: The Dementia In Residential care: EduCation intervention Trial (DIRECT was conducted to determine if delivery of education designed to meet the perceived need of GPs and care staff improves the quality of life of participants with dementia living in residential care. METHODOLOGY/PRINCIPAL FINDINGS: This cluster-randomised controlled trial was conducted in 39 residential aged care facilities in the metropolitan area of Perth, Western Australia. 351 care facility residents aged 65 years and older with Mini-Mental State Examination ≤ 24, their GPs and facility staff participated. Flexible education designed to meet the perceived needs of learners was delivered to GPs and care facility staff in intervention groups. The primary outcome of the study was self-rated quality of life of participants with dementia, measured using the QOL-Alzheimer's Disease Scale (QOL-AD at 4 weeks and 6 months after the conclusion of the intervention. Analysis accounted for the effect of clustering by using multi-level regression analysis. Education of GPs or care facility staff did not affect the primary outcome at either 4 weeks or 6 months. In a post hoc analysis excluding facilities in which fewer than 50% of staff attended an education session, self-rated QOL-AD scores were 6.14 points (adjusted 95%CI 1.14, 11.15 higher at four-week follow-up among residents in facilities randomly assigned to the education intervention. CONCLUSION: The education intervention directed at care facilities or GPs did not improve the quality of life ratings of participants with dementia as a group. This may be explained by the poor adherence to the intervention programme, as participants with dementia living in facilities where staff participated at least minimally seemed to benefit. TRIAL REGISTRATION: ANZCTR.org.au ACTRN12607000417482.

  2. Effects of host cell sterol composition upon internalization ofYersinia pseudotuberculosisand clustered β1 integrin.

    Science.gov (United States)

    Kim, JiHyun; Fukuto, Hana S; Brown, Deborah A; Bliska, James B; London, Erwin

    2018-01-26

    Yersinia pseudotuberculosis is a foodborne pathogenic bacterium that causes acute gastrointestinal illness, but its mechanisms of infection are incompletely described. We examined how host cell sterol composition affected Y. pseudotuberculosis uptake. To do this, we depleted or substituted cholesterol in human MDA-MB-231 epithelial cells with various alternative sterols. Decreasing host cell cholesterol significantly reduced pathogen internalization. When host cell cholesterol was substituted with various sterols, only desmosterol and 7-dehydrocholesterol supported internalization. This specificity was not due to sterol dependence of bacterial attachment to host cells, which was similar with all sterols studied. Because a key step in Y. pseudotuberculosis internalization is interaction of the bacterial adhesins invasin and YadA with host cell β1 integrin, we compared the sterol dependence of wildtype Y. pseudotuberculosis internalization with that of Δ inv , Δ yadA , and Δ inv Δ yadA mutant strains. YadA deletion decreased bacterial adherence to host cells, whereas invasin deletion had no effect. Nevertheless, host cell sterol substitution had a similar effect on internalization of these bacterial deletion strains as on the wildtype bacteria. The Δ inv Δ yadA double mutant adhered least to cells and so was not significantly internalized. The sterol structure dependence of Y. pseudotuberculosis internalization differed from that of endocytosis, as monitored using antibody-clustered β1 integrin and previous studies on other proteins, which had a more permissive sterol dependence. This study suggests that agents could be designed to interfere with internalization of Yersinia without disturbing endocytosis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. A high fat diet containing saturated but not unsaturated fatty acids enhances T cell receptor clustering on the nanoscale.

    Science.gov (United States)

    Shaikh, Saame Raza; Boyle, Sarah; Edidin, Michael

    2015-09-01

    Cell culture studies show that the nanoscale lateral organization of surface receptors, their clustering or dispersion, can be altered by changing the lipid composition of the membrane bilayer. However, little is known about similar changes in vivo, which can be effected by changing dietary lipids. We describe the use of a newly developed method, k-space image correlation spectroscopy, kICS, for analysis of quantum dot fluorescence to show that a high fat diet can alter the nanometer-scale clustering of the murine T cell receptor, TCR, on the surface of naive CD4(+) T cells. We found that diets enriched primarily in saturated fatty acids increased TCR nanoscale clustering to a level usually seen only on activated cells. Diets enriched in monounsaturated or n-3 polyunsaturated fatty acids had no effect on TCR clustering. Also none of the high fat diets affected TCR clustering on the micrometer scale. Furthermore, the effect of the diets was similar in young and middle aged mice. Our data establish proof-of-principle that TCR nanoscale clustering is sensitive to the composition of dietary fat. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. 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.

  5. 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

  6. 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.

  7. Optical Imaging of Cells with Gold Nanoparticle Clusters as Light Scattering Contrast Agents

    DEFF Research Database (Denmark)

    Tanev, Stoyan

    2011-01-01

    This chapter has two main objectives. First, to review a number of examples illustrating the application of the FDTD approach to the modeling of some typical light scattering configurations that could be associated with flow cytometry. Second, to provide a thorough discussion of these new...... developments in advanced cytometry research by pointing out potential new research directions. A brief description of the FDTD method focusing on the features associated with its application to modeling of light scattering and OPCM cell imaging experiments is provided. The examples include light scattering...... from OPCM imaging of single biological cells in conditions of controlled refractive index matching (RIM) and labeling by diffused and clustered gold NPs. The chapter concludes with a discussion and suggestions for future research....

  8. Multi-color fluorescence imaging of sub-cellular dynamics of cancer cells in live mice

    Science.gov (United States)

    Hoffman, Robert M.

    2006-02-01

    We have genetically engineered dual-color fluorescent cells with one color in the nucleus and the other in the cytoplasm that enables real-time nuclear-cytoplasmic dynamics to be visualized in living cells in the cytoplasm in vivo as well as in vitro. To obtain the dual-color cells, red fluorescent protein (RFP) was expressed of the cancer cells, and green fluorescent protein (GFP) linked to histone H2B was expressed in the nucleus. Mitotic cells were visualized by whole-body imaging after injection in the mouse ear. Common carotid artery or heart injection of dual-color cells and a reversible skin flap enabled the external visualization of the dual-color cells in microvessels in the mouse where extreme elongation of the cell body as well as the nucleus occurred. The migration velocities of the dual-color cancer cells in the capillaries were measured by capturing individual images of the dual-color fluorescent cells over time. Human HCT-116-GFP-RFP colon cancer and mouse mammary tumor (MMT)-GFP-RFP cells were injected in the portal vein of nude mice. Extensive clasmocytosis (destruction of the cytoplasm) of the HCT-116-GFP-RFP cells occurred within 6 hours. The data suggest rapid death of HCT-116-GFP-RFP cells in the portal vein. In contrast, MMT-GFP-RFP cells injected into the portal vein mostly survived and formed colonies in the liver. However, when the host mice were pretreated with cyclophosphamide, the HCT-116-GFP-RFP cells also survived and formed colonies in the liver after portal vein injection. These results suggest that a cyclophosphamide-sensitive host cellular system attacked the HCT-116-GFP-RFP cells but could not effectively kill the MMT-GFP-RFP cells. With the ability to continuously image cancer cells at the subcellular level in the live animal, our understanding of the complex steps of metastasis will significantly increase. In addition, new drugs can be developed to target these newly visible steps of metastasis.

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

    Science.gov (United States)

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

    2014-11-01

    The "stem cell" has become arguably one of the most important biological tools in the arsenal of translational research directed at regeneration and repair. It remains to be seen whether every tissue has its own stem cell niche, although relatively recently a large amount of research has focused on isolating and characterizing tissue-specific stem cell populations, as well as those that are able to be directed to transdifferentiate into a variety of different lineages. Traditionally, stem cells are isolated from the viable tissue of embryonic, fetal, or adult living hosts; from "fresh" donated tissues that have been surgically or otherwise removed (biopsies), or obtained directly from tissues within minutes to several hours post mortem (PM). These human progenitor/stem cell sources remain potentially highly controversial, since they are accompanied by various still-unresolved ethical, social, moral and legal challenges. Due to the limited number of "live" donors, the small amount of material obtained from biopsies and difficulties during purification processes, harvesting from cadaveric material presents itself as an alternative strategy that could provide a hitherto untapped source of stem cells. However, PM stem cells are not without their own unique set of limitations including difficulty of obtaining samples, limited supply of material, variations in delay between death and sample collection, possible lack of medication history and suboptimal retrospective assignment of diagnostic and demographic data. This article is part of a Directed Issue entitled: Regenerative Medicine: The challenge of translation. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Adolescents' experiences of living with sickle cell disease: An integrative narrative review of the literature.

    Science.gov (United States)

    Poku, Brenda Agyeiwaa; Caress, Ann-Louise; Kirk, Susan

    2018-04-01

    Sickle Cell Disease is the commonest monogenic haemoglobinopathy worldwide. Living with a long-term condition such as sickle cell disease during adolescence constitutes a significant challenge for the key stakeholders due to the combined effects of chronic illness and adolescent development. For adolescents with sickle cell disease to be cared for and supported appropriately and effectively, it is crucial that health professionals have a comprehensive knowledge and understanding of how adolescents experience living with the condition. While there is developing literature about how adolescent's experience sickle cell disease, this body of research has not been critically reviewed and synthesised. To identify, critically appraise and synthesise primary research exploring adolescents' experiences of living with sickle cell disease to make recommendations for practice and research. Integrative narrative review. A systematic search of 10 electronic databases and key journals was conducted to identify studies from the inception of databases to September 2016. Inclusion criteria: adolescents with sickle cell disease aged 12-19 years, primary data on adolescents' own perspectives, and published in English. Data were extracted on study contexts, methodology/design, theoretical constructs, participants, and key findings. Findings from included studies were synthesised using the integrative narrative approach. Additionally, the methodological quality of studies was assessed using the Hawker et al. (2002) appraisal checklist. 683 studies were identified, of which 40 fulfilled the inclusion criteria. Nine broad themes emerged: knowledge and understanding of the condition, symptom experiences, self-management, attitude to treatment, healthcare experiences, social relationships, difference and striving for normality, school experiences, and emotional well-being and coping. Majority of the studies were of moderate quality methodologically. Quality assessment demonstrated a high

  11. Both Laminin and Schwann cell Dystroglycan are necessary for proper clustering of Sodium Channels at Nodes of Ranvier.

    Science.gov (United States)

    Occhi, S.; Zambroni, D.; Del Carro, U.; Amadio, S.; Sirkowski, E. E.; Scherer, SS.; Campbell, K.; Moore, S.A.; Chen, Z-L.; Strickland, S.; Di Muzio, A.; Uncini, A.; Wrabetz, L.; Feltri, ML.

    2005-01-01

    Nodes of Ranvier are specialized axonal domains, where voltage-gated sodium channels cluster. How axons cluster molecules in discrete domains is largely unknown. Both axons and glia probably provide constraining mechanisms that contribute to domain formation. Proper sodium channel clustering in peripheral nerves depends on contact from Schwann cell microvilli, where at least one molecule, gliomedin, binds the sodium channel complex and induces its clustering. Furthermore, mice lacking Schwann cell dystroglycan have aberrant microvilli and poorly clustered sodium channels. Dystroglycan could interact at the basal lamina or at the axon-glial surface. Since dystroglycan is a laminin receptor, and laminin-2 mutations (merosin-deficient-congenital-muscular-dystrophy, MDC1A) cause reduced nerve conduction velocity, we asked whether laminins are involved. Here we show that the composition of both laminins and the dystroglycan complex at nodes differs from that of internodes. Mice defective in laminin-2 have poorly formed microvilli and abnormal sodium clusters. These abnormalities are similar, albeit less severe, than those of mice lacking dystroglycan. However, mice lacking all Schwann cell laminins show severe nodal abnormalities, suggesting other laminins compensate for the lack of laminin 2. Thus, even though laminins are located at a distance from the axo-glial junction, they are required for proper clustering of sodium channels. Laminins, through their specific nodal receptors and cytoskeletal linkages, may participate in the formation of mechanisms that constrain clusters at nodes. Finally, abnormal sodium channel clusters are present in a patient with MDC1A, providing a molecular basis for the reduced nerve conduction velocity in this disorder. PMID:16221851

  12. Evolution of long-lived globular cluster stars. III. Effect of the initial helium spread on the position of stars in a synthetic Hertzsprung-Russell diagram

    Science.gov (United States)

    Chantereau, W.; Charbonnel, C.; Meynet, G.

    2016-08-01

    Context. Globular clusters host multiple populations of long-lived low-mass stars whose origin remains an open question. Several scenarios have been proposed to explain the associated photometric and spectroscopic peculiarities. They differ, for instance, in the maximum helium enrichment they predict for stars of the second population, which these stars can inherit at birth as the result of the internal pollution of the cluster by different types of stars of the first population. Aims: We present the distribution of helium-rich stars in present-day globular clusters as it is expected in the original framework of the fast-rotating massive stars scenario (FRMS) as first-population polluters. We focus on NGC 6752. Methods: We completed a grid of 330 stellar evolution models for globular cluster low-mass stars computed with different initial chemical compositions corresponding to the predictions of the original FRMS scenario for [Fe/H] = -1.75. Starting from the initial helium-sodium relation that allows reproducing the currently observed distribution of sodium in NGC 6752, we deduce the helium distribution expected in that cluster at ages equal to 9 and 13 Gyr. We distinguish the stars that are moderately enriched in helium from those that are very helium-rich (initial helium mass fraction below and above 0.4, respectively), and compare the predictions of the FRMS framework with other scenarios for globular cluster enrichment. Results: The effect of helium enrichment on the stellar lifetime and evolution reduces the total number of very helium-rich stars that remain in the cluster at 9 and 13 Gyr to only 12% and 10%, respectively, from an initial fraction of 21%. Within this age range, most of the stars still burn their hydrogen in their core, which widens the MS band significantly in effective temperature. The fraction of very helium-rich stars drops in the more advanced evolution phases, where the associated spread in effective temperature strongly decreases. These

  13. Realizing 'integral control' in living cells: how to overcome leaky integration due to dilution?

    Science.gov (United States)

    Qian, Yili; Del Vecchio, Domitilla

    2018-02-01

    A major problem in the design of synthetic genetic circuits is robustness to perturbations and uncertainty. Because of this, there have been significant efforts in recent years in finding approaches to implement integral control in genetic circuits. Integral controllers have the unique ability to make the output of a process adapt perfectly to disturbances. However, implementing an integral controller is challenging in living cells. This is because a key aspect of any integral controller is a 'memory' element that stores the accumulation (integral) of the error between the output and its desired set-point. The ability to realize such a memory element in living cells is fundamentally challenged by the fact that all biomolecules dilute as cells grow, resulting in a 'leaky' memory that gradually fades away. As a consequence, the adaptation property is lost. Here, we propose a general principle for designing integral controllers such that the performance is practically unaffected by dilution. In particular, we mathematically prove that if the reactions implementing the integral controller are all much faster than dilution, then the adaptation error due to integration leakiness becomes negligible. We exemplify this design principle with two synthetic genetic circuits aimed at reaching adaptation of gene expression to fluctuations in cellular resources. Our results provide concrete guidance on the biomolecular processes that are most appropriate for implementing integral controllers in living cells. © 2018 The Author(s).

  14. Inter-chromosomal Contact Properties in Live-Cell Imaging and in Hi-C.

    Science.gov (United States)

    Maass, Philipp G; Barutcu, A Rasim; Weiner, Catherine L; Rinn, John L

    2018-03-15

    Imaging (fluorescence in situ hybridization [FISH]) and genome-wide chromosome conformation capture (Hi-C) are two major approaches to the study of higher-order genome organization in the nucleus. Intra-chromosomal and inter-chromosomal interactions (referred to as non-homologous chromosomal contacts [NHCCs]) have been observed by several FISH-based studies, but locus-specific NHCCs have not been detected by Hi-C. Due to crosslinking, neither of these approaches assesses spatiotemporal properties. Toward resolving the discrepancies between imaging and Hi-C, we sought to understand the spatiotemporal properties of NHCCs in living cells by CRISPR/Cas9 live-cell imaging (CLING). In mammalian cells, we find that NHCCs are stable and occur as frequently as intra-chromosomal interactions, but NHCCs occur at farther spatial distance that could explain their lack of detection in Hi-C. By revealing the spatiotemporal properties in living cells, our study provides fundamental insights into the biology of NHCCs. Copyright © 2018 Elsevier Inc. All rights reserved.

  15. Hemi-fused structure mediates and controls fusion and fission in live cells.

    Science.gov (United States)

    Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul; Wen, Peter J; Krystofiak, Evan S; Villarreal, Seth A; Chiang, Hsueh-Cheng; Kachar, Bechara; Wu, Ling-Gang

    2016-06-23

    Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission.

  16. Broccoli-shaped biosensor hierarchy for electrochemical screening of noradrenaline in living cells.

    Science.gov (United States)

    Emran, Mohammed Y; Mekawy, Moataz; Akhtar, Naeem; Shenashen, Mohamed A; El-Sewify, Islam M; Faheem, Ahmed; El-Safty, Sherif A

    2018-02-15

    Monitoring and determination of ultra-trace concentrations of monoamine neurotransmitter such as noradrenaline (NA) in living cells with simple, sensitive and selective assays are significantly interesting. We design NA-electrode sensing system based on C-, N-doped NiO broccoli-like hierarchy (CNNB). The spherical broccoli-head umbrella architectures associated with nano-tubular arrangements enabled to tailor NA biosensor design. The homogenous doping and anisotropic dispersion of CN nanospheres along the entire NB head nanotubes lead to creating of abundant electroactive sites in the interior tubular vessels and outer surfaces for ultrasensitive detection of NA in living cells such as PC12. The CNNB biosensor electrodes showed efficient electrocatalytic activity, enhanced kinetics for electrooxidation of NA, and fast electron-transfer between electrode-electrolyte interface surfaces, enabling synergistic enhancement in sensitivity, and selectivity at a low-detectable concentration of ∼ 6nM and reproducibility of broccoli-shaped NA-electrodes. The integrated CNNB biosensor electrodes showed evidence of monitoring and screening of NA released from PC12 cells under K + ion-extracellular stimulation process. The unique features of CNNB in terms of NA-selectivity among multi-competitive components, long-term stability during the detection of NA may open their practical, in-vitro application for extracellular monoamine neurotransmitters detection in living cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell.

    Science.gov (United States)

    Hein, Birka; Willig, Katrin I; Hell, Stefan W

    2008-09-23

    We demonstrate far-field optical imaging with subdiffraction resolution of the endoplasmic reticulum (ER) in the interior of a living mammalian cell. The diffraction barrier is overcome by applying stimulated emission depletion (STED) on a yellow fluorescent protein tag. Imaging individual structural elements of the ER revealed a focal plane (x, y) resolution of <50 nm inside the living cell, corresponding to a 4-fold improvement over that of a confocal microscope and a 16-fold reduction in the focal-spot cross-sectional area. A similar gain in resolution is realized with both pulsed- and continuous-wave laser illumination. Images of highly convoluted parts of the ER reveal a similar resolution improvement in 3D optical sectioning by a factor of 3 along the optic axis (z). Time-lapse STED recordings document morphological changes of the ER over time. Thus, nanoscale 3D imaging of organelles in the interior of living cells greatly expands the scope of light microscopy in cell biology.

  18. Neutron scattering to study membrane systems: from lipid vesicles to living cells.

    Energy Technology Data Exchange (ETDEWEB)

    Nickels, Jonathan D. [ORNL; Chatterjee, Sneha [ORNL; Stanley, Christopher B. [ORNL; Qian, Shuo [ORNL; Cheng, Xiaolin [ORNL; Myles, Dean A A [ORNL; Standaert, Robert F. [ORNL; Elkins, James G. [ORNL; Katsaras, John [ORNL

    2017-03-01

    The existence and role of lateral lipid organization in biological membranes has been studied and contested for more than 30 years. Lipid domains, or rafts, are hypothesized as scalable compartments in biological membranes, providing appropriate physical environments to their resident membrane proteins. This implies that lateral lipid organization is associated with a range of biological functions, such as protein co-localization, membrane trafficking, and cell signaling, to name just a few. Neutron scattering techniques have proven to be an excellent tool to investigate these structural features in model lipids, and more recently, in living cells. I will discuss our recent work using neutrons to probe the structure and mechanical properties in model lipid systems and our current efforts in using neutrons to probe the structure and organization of the bilayer in a living cell. These efforts in living cells have used genetic and biochemical strategies to generate a large neutron scattering contrast, making the membrane visible. I will present our results showing in vivo bilayer structure and discuss the outlook for this approach.

  19. 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.

  20. Imaging of native transcription factors and histone phosphorylation at high resolution in live cells.

    Science.gov (United States)

    Conic, Sascha; Desplancq, Dominique; Ferrand, Alexia; Fischer, Veronique; Heyer, Vincent; Reina San Martin, Bernardo; Pontabry, Julien; Oulad-Abdelghani, Mustapha; Babu N, Kishore; Wright, Graham D; Molina, Nacho; Weiss, Etienne; Tora, László

    2018-04-02

    Fluorescent labeling of endogenous proteins for live-cell imaging without exogenous expression of tagged proteins or genetic manipulations has not been routinely possible. We describe a simple versatile antibody-based imaging approach (VANIMA) for the precise localization and tracking of endogenous nuclear factors. Our protocol can be implemented in every laboratory allowing the efficient and nonharmful delivery of organic dye-conjugated antibodies, or antibody fragments, into different metazoan cell types. Live-cell imaging permits following the labeled probes bound to their endogenous targets. By using conventional and super-resolution imaging we show dynamic changes in the distribution of several nuclear transcription factors (i.e., RNA polymerase II or TAF10), and specific phosphorylated histones (γH2AX), upon distinct biological stimuli at the nanometer scale. Hence, considering the large panel of available antibodies and the simplicity of their implementation, VANIMA can be used to uncover novel biological information based on the dynamic behavior of transcription factors or posttranslational modifications in the nucleus of single live cells. © 2018 Conic et al.

  1. Single-molecule live-cell imaging of bacterial DNA repair and damage tolerance.

    Science.gov (United States)

    Ghodke, Harshad; Ho, Han; van Oijen, Antoine M

    2018-02-19

    Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow individual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA-repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA-repair processes in prokaryotes. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  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. Fabrication of micropatterned arrays of gold nanoparticles for photothermal manipulation of living cells.

    Science.gov (United States)

    Polleux, Julien; Baffou, Guillaume

    2014-01-01

    The fabrication of micro/nanostructured surfaces functionalized with stimulus-responsive chemical groups proved to be an interesting approach to simultaneously confine cell adhesion and manipulate cell-substrate interactions down to the single cell level. However, reversibility of stimulus-triggered systems is often not possible or exhibits slow switching kinetics. In contrast to such setups, gold nanoparticles have the properties to efficiently and reversibly generate heat under illumination at their plasmon resonance band. Thus, photo-induced heating could be used to directly and locally interface living cells and dynamically tailor the interactions to their adhesive environment. In the present chapter, we will first detail the preparation of micropatterned and functionalized gold nanoparticles immobilized on glass coverslips, and then report how to reliably characterize the photothermal properties of such substrates that enable the dynamic manipulation of cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. IncucyteDRC: An R package for the dose response analysis of live cell imaging data

    Science.gov (United States)

    Chapman, Philip J.; James, Dominic I.; Watson, Amanda J.; Hopkins, Gemma V.; Waddell, Ian D.; Ogilvie, Donald J.

    2016-01-01

    We present IncucyteDRC, an R package for the analysis of data from live cell imaging cell proliferation experiments carried out on the Essen Biosciences IncuCyte ZOOM instrument. The package provides a simple workflow for summarising data into a form that can be used to calculate dose response curves and EC50 values for small molecule inhibitors. Data from different cell lines, or cell lines grown under different conditions, can be normalised as to their doubling time. A simple graphical web interface, implemented using shiny, is provided for the benefit of non-R users. The software is potentially useful to any research group studying the impact of small molecule inhibitors on cell proliferation using the IncuCyte ZOOM. PMID:27703665

  5. 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.

  6. High content live cell imaging for the discovery of new antimalarial marine natural products.

    Science.gov (United States)

    Cervantes, Serena; Stout, Paige E; Prudhomme, Jacques; Engel, Sebastian; Bruton, Matthew; Cervantes, Michael; Carter, David; Tae-Chang, Young; Hay, Mark E; Aalbersberg, William; Kubanek, Julia; Le Roch, Karine G

    2012-01-03

    The human malaria parasite remains a burden in developing nations. It is responsible for up to one million deaths a year, a number that could rise due to increasing multi-drug resistance to all antimalarial drugs currently available. Therefore, there is an urgent need for the discovery of new drug therapies. Recently, our laboratory developed a simple one-step fluorescence-based live cell-imaging assay to integrate the complex biology of the human malaria parasite into drug discovery. Here we used our newly developed live cell-imaging platform to discover novel marine natural products and their cellular phenotypic effects against the most lethal malaria parasite, Plasmodium falciparum. A high content live cell imaging platform was used to screen marine extracts effects on malaria. Parasites were grown in vitro in the presence of extracts, stained with RNA sensitive dye, and imaged at timed intervals with the BD Pathway HT automated confocal microscope. Image analysis validated our new methodology at a larger scale level and revealed potential antimalarial activity of selected extracts with a minimal cytotoxic effect on host red blood cells. To further validate our assay, we investigated parasite's phenotypes when incubated with the purified bioactive natural product bromophycolide A. We show that bromophycolide A has a strong and specific morphological effect on parasites, similar to the ones observed from the initial extracts. Collectively, our results show that high-content live cell-imaging (HCLCI) can be used to screen chemical libraries and identify parasite specific inhibitors with limited host cytotoxic effects. All together we provide new leads for the discovery of novel antimalarials. © 2011 Cervantes et al; licensee BioMed Central Ltd.

  7. 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)

  8. 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 subcellular localization of neuronal membrane signaling molecules such as receptors and ion channels depends on intracellular trafficking mechanisms. Essentially, vesicular trafficking mechanisms ensure that a large number of membrane proteins are correctly targeted to different subcellular...... 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...

  9. Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation

    Science.gov (United States)

    Sutherland, B. M.; Bennett, P. V.; Sidorkina, O.; Laval, J.; Lowenstein, D. I. (Principal Investigator)

    2000-01-01

    Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

  10. Femtosecond laser printing of living cells using absorbing film-assisted laser-induced forward transfer

    Science.gov (United States)

    Hopp, Béla; Smausz, Tomi; Szabó, Gábor; Kolozsvári, Lajos; Kafetzopoulos, Dimitris; Fotakis, Costas; Nógrádi, Antal

    2012-01-01

    The applicability of a femtosecond KrF laser in absorbing film-assisted, laser-induced forward transfer of living cells was studied. The absorbing materials were 50-nm-thick metal films and biomaterials (gelatine, Matrigel, each 50 μm thick, and polyhydroxybutyrate, 2 μm). The used cell types were human neuroblastoma, chronic myeloid leukemia, and osteogenic sarcoma cell lines, and primary astroglial rat cells. Pulses of a 500-fs KrF excimer laser focused onto the absorbing layer in a 250-μm diameter spot with 225 mJ/cm2 fluence were used to transfer the cells to the acceptor plate placed at 0.6 mm distance, which was a glass slide either pure or covered with biomaterials. While the low-absorptivity biomaterial absorbing layers proved to be ineffective in transfer of cells, when applied on the surface of acceptor plate, the wet gelatine and Matrigel layers successfully ameliorated the impact of the cells, which otherwise did not survive the arrival onto a hard surface. The best short- and long-term survival rate was between 65% and 70% for neuroblastoma and astroglial cells. The long-term survival of the transferred osteosarcoma cells was low, while the myeloid leukemia cells did not tolerate the procedure under the applied experimental conditions.

  11. Structural and Functional Characterization of Human Stem-Cell-Derived Retinal Organoids by Live Imaging.

    Science.gov (United States)

    Browne, Andrew W; Arnesano, Cosimo; Harutyunyan, Narine; Khuu, Thien; Martinez, Juan Carlos; Pollack, Harvey A; Koos, David S; Lee, Thomas C; Fraser, Scott E; Moats, Rex A; Aparicio, Jennifer G; Cobrinik, David

    2017-07-01

    Human pluripotent stem cell (hPSC)-derived retinal organoids are a platform for investigating retinal development, pathophysiology, and cellular therapies. In contrast to histologic analysis in which multiple specimens fixed at different times are used to reconstruct developmental processes, repeated analysis of the same living organoids provides a more direct means to characterize changes. New live imaging modalities can provide insights into retinal organoid structure and metabolic function during in vitro growth. This study employed live tissue imaging to characterize retinal organoid development, including metabolic changes accompanying photoreceptor differentiation. Live hPSC-derived retinal organoids at different developmental stages were examined for microanatomic organization and metabolic function by phase contrast microscopy, optical coherence tomography (OCT), fluorescence lifetime imaging microscopy (FLIM), and hyperspectral imaging (HSpec). Features were compared to those revealed by histologic staining, immunostaining, and microcomputed tomography (micro-CT) of fixed organoid tissue. We used FLIM and HSpec to detect changes in metabolic activity as organoids differentiated into organized lamellae. FLIM detected increased glycolytic activity and HSpec detected retinol and retinoic acid accumulation in the organoid outer layer, coinciding with photoreceptor genesis. OCT enabled imaging of lamellae formed during organoid maturation. Micro-CT revealed three-dimensional structure, but failed to detect lamellae. Live imaging modalities facilitate real-time and nondestructive imaging of retinal organoids as they organize into lamellar structures. FLIM and HSpec enable rapid detection of lamellar structure and photoreceptor metabolism. Live imaging techniques may aid in the continuous evaluation of retinal organoid development in diverse experimental and cell therapy settings.

  12. Spatial scales of living cells and their energetic and informational capacity.

    Science.gov (United States)

    Bormashenko, Edward; Voronel, Alexander

    2017-12-04

    Physical (thermodynamic and kinetic), chemical, and biological reasoning restrict the spatial dimensions of living cells (prokaryotic and eukaryotic) and confine them to between 1 and 100 µm. Cells should necessarily be macroscopic, dissipative objects, resisting thermal fluctuations and providing sufficient informational capacity. The upper limit of the spatial dimensions of cells is supplied by their ability to withstand gravity and inertia forces under reasonable deformations. The upper limit of cell dimensions is also governed by the hierarchy of characteristic time scales, inherent for mass and heat transport. For micron-scaled cells, the "traffic time" (namely a typical time necessary for the migration of one enzyme to another) is on the order of magnitude of a millisecond, which coincides with the characteristic time scale of a single round of the catalytic enzyme cycle. The macroscopic dimensions of living cells (seen as dissipative systems) and the hierarchy of time scales of the mass transfer processes vs. those inherent for heat transport and viscous dissipation give rise to the irreversibility of biological processes.

  13. Quantitative electrophysiological monitoring of anti-histamine drug effects on live cells via reusable sensor platforms.

    Science.gov (United States)

    Pham Ba, Viet Anh; Cho, Dong-Guk; Kim, Daesan; Yoo, Haneul; Ta, Van-Thao; Hong, Seunghun

    2017-08-15

    We demonstrated the quantitative electrophysiological monitoring of histamine and anti-histamine drug effects on live cells via reusable sensor platforms based on carbon nanotube transistors. This method enabled us to monitor the real-time electrophysiological responses of a single HeLa cell to histamine with different concentrations. The measured electrophysiological responses were attributed to the activity of histamine type 1 receptors on a HeLa cell membrane by histamine. Furthermore, the effects of anti-histamine drugs such as cetirizine or chlorphenamine on the electrophysiological activities of HeLa cells were also evaluated quantitatively. Significantly, we utilized only a single device to monitor the responses of multiple HeLa cells to each drug, which allowed us to quantitatively analyze the antihistamine drug effects on live cells without errors from the device-to-device variation in device characteristics. Such quantitative evaluation capability of our method would promise versatile applications such as drug screening and nanoscale bio sensor researches. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Intracellular stress tomography reveals stress focusing and structural anisotropy in cytoskeleton of living cells

    Science.gov (United States)

    Hu, Shaohua; Chen, Jianxin; Fabry, Ben; Numaguchi, Yasushi; Gouldstone, Andrew; Ingber, Donald E.; Fredberg, Jeffrey J.; Butler, James P.; Wang, Ning

    2003-01-01

    We describe a novel synchronous detection approach to map the transmission of mechanical stresses within the cytoplasm of an adherent cell. Using fluorescent protein-labeled mitochondria or cytoskeletal components as fiducial markers, we measured displacements and computed stresses in the cytoskeleton of a living cell plated on extracellular matrix molecules that arise in response to a small, external localized oscillatory load applied to transmembrane receptors on the apical cell surface. Induced synchronous displacements, stresses, and phase lags were found to be concentrated at sites quite remote from the localized load and were modulated by the preexisting tensile stress (prestress) in the cytoskeleton. Stresses applied at the apical surface also resulted in displacements of focal adhesion sites at the cell base. Cytoskeletal anisotropy was revealed by differential phase lags in X vs. Y directions. Displacements and stresses in the cytoskeleton of a cell plated on poly-L-lysine decayed quickly and were not concentrated at remote sites. These data indicate that mechanical forces are transferred across discrete cytoskeletal elements over long distances through the cytoplasm in the living adherent cell.

  15. Real time imaging of mRNA expression dynamics in live cells using protein complementation methods

    Science.gov (United States)

    Meller, Amit

    2009-03-01

    Traditional methods for mRNA quantification in cells, such as northern blots, quantitative PCR or microarrays assays, require cell lysis and therefore do not preserve its dynamics. These methods cannot be used to probe the spatio-temporal localization of mRNA in cells, which provide useful information for a wide range biomolecular process, including RNA metabolizim, expression kinetics and RNA interference. To probe mRNA dynamics in live prokaryotic and eukaryotic cells, we develop a method, which exploit the strong affinity of the eukaryotic initiation factor 4A (eIF4A) to specific RNA aptamers. Two parts of the eIF4A are fused to a split Green Fluorescence Protein (GFP), and are expressed in the cells at high abundance. However, only when the RNA apatmer is also present, the two protein parts complement and become fluorescent. Thus, the fluorescent background remains low, allowing us to directly image the expression of mRNA molecules in live e-coli cells from its early onset, over hours. We find that the expression kinetics can be classified in one out of at least three forms, which also display distinct spatial distributions. I will discuss the possible biological origin for these distributions and their time evolution.

  16. Real-time detecting gelatinases activity in living cells by FRET imaging

    Science.gov (United States)

    Yang, Jie; Zhang, Zhihong; Liu, Bifeng; Luo, Qingming

    2006-01-01

    Degradation of the extracellular matrix by Matrix metalloproteinases (MMPs) not only enhances tumor invasion, but also affects tumor cell behaviour and leads to cancer progression. To monitor gelatinases (contain MMP2 and MMP9) activity in living cells, we constructed a vector that encoded a gelatinases recognition site (GRS) between citrine (mutation of EYFP Q69M) in N terminal and ECFP in C terminal. Because Gelatinases are secretory proteins and act outside of cell, an expressing vector displayed the fusion protein on cellular surface was used for this FRET gene probe. On expression of YFP-GRS-ECFP in MCF-7 cells that expressed no gelatinases, we were able to observe the efficient transfer of energy from excited ECFP to YFP within the YFP-GRS-ECFP molecule. However, the fusion protein YFP-GRS-ECFP was expressed in MDA-MB 453s cell line with high secretory gelatinases, so YFP-GRS-ECFP was cleaved by gelatinases, no such transfer of energy was detected and fluorescence signal disappeared in YFP channel since YFP protein was cut down. Moreover, Doxycycline, a MMP inhibitor, could make FRET signal increase and fluorescence signal appeared in YFP channel. Thus, the FRET probe YFP-GRS-ECFP can sensitively and reliably monitor gelatinases activation in living cells and can be used for screening MMP inhibitors.

  17. Size and dynamics of caveolae studied using nanoparticles in living endothelial cells.

    Science.gov (United States)

    Wang, Zhenjia; Tiruppathi, Chinnaswamy; Minshall, Richard D; Malik, Asrar B

    2009-12-22

    Caveolae are plasma membrane invaginations prominent in all endothelial cells lining blood vessels. Caveolae characteristically bud to form free cytoplasmic vesicles capable of transporting carrier proteins such as albumin through the cell. However, caveolae size distribution and dynamics in living endothelial cells and ability of caveolae to internalize nanoparticles are not well understood. We demonstrate here the design of a dual-color nanoparticle pair to measure noninvasively caveolae size and dynamics. First, we coated nanoparticles with BSA (bovine serum albumin) to address whether albumin promoted their delivery. Albumin has been shown to bind to protein on endothelial cell surface localized in caveolae and activate albumin endocytosis. Imaging of BSA-coated nanoparticles varying from 20 to 100 nm in diameter in endothelial cells demonstrated that caveolae-mediated nanoparticle uptake was dependent on albumin coating of particles. We also showed that caveolae could accommodate up to 100 nm diameter nanoparticles, a size larger than the diameter of typical caveolae, suggesting compliant property of caveolae. Together, our results show the feasibility of tracking multicolored nanoparticles in living endothelial cells and potential usefulness for designing therapeutic nanoparticle cargo to cross the limiting vessel wall endothelial barrier.

  18. Novel Real-Time Proximity Assay for Characterizing Multiple Receptor Interactions on Living Cells.

    Science.gov (United States)

    Bondza, Sina; Björkelund, Hanna; Nestor, Marika; Andersson, Karl; Buijs, Jos

    2017-12-19

    Cellular receptor activity is often controlled through complex mechanisms involving interactions with multiple molecules, which can be soluble ligands and/or other cell surface molecules. In this study, we combine a fluorescence-based technology for real-time interaction analysis with fluorescence quenching to create a novel time-resolved proximity assay to study protein-receptor interactions on living cells. This assay extracts the binding kinetics and affinity for two proteins if they bind in proximity on the cell surface. One application of real-time proximity interaction analysis is to study relative levels of receptor dimerization. The method was primarily evaluated using the HER2 binding antibodies Trastuzumab and Pertuzumab and two EGFR binding antibodies including Cetuximab. Using Cetuximab and Trastuzumab, proximity of EGFR and HER2 was investigated before and after treatment of cells with the tyrosine-kinase inhibitor Gefitinib. Treated cells displayed 50% increased proximity signal, whereas the binding characteristics of the two antibodies were not significantly affected, implying an increase in the EGFR-HER2 dimer level. These results demonstrate that real-time proximity interaction analysis enables determination of the interaction rate constants and affinity of two ligands while simultaneously quantifying their relative colocalization on living cells.

  19. Recent Developments of the Local Effect Model (LEM) - Implications of clustered damage on cell transformation

    Science.gov (United States)

    Elsässer, Thilo

    Exposure to radiation of high-energy and highly charged ions (HZE) causes a major risk to human beings, since in long term space explorations about 10 protons per month and about one HZE particle per month hit each cell nucleus (1). Despite the larger number of light ions, the high ionisation power of HZE particles and its corresponding more complex damage represents a major hazard for astronauts. Therefore, in order to get a reasonable risk estimate, it is necessary to take into account the entire mixed radiation field. Frequently, neoplastic cell transformation serves as an indicator for the oncogenic potential of radiation exposure. It can be measured for a small number of ion and energy combinations. However, due to the complexity of the radiation field it is necessary to know the contribution to the radiation damage of each ion species for the entire range of energies. Therefore, a model is required which transfers the few experimental data to other particles with different LETs. We use the Local Effect Model (LEM) (2) with its cluster extension (3) to calculate the relative biological effectiveness (RBE) of neoplastic transformation. It was originally developed in the framework of hadrontherapy and is applicable for a large range of ions and energies. The input parameters for the model include the linear-quadratic parameters for the induction of lethal events as well as for the induction of transformation events per surviving cell. Both processes of cell inactivation and neoplastic transformation per viable cell are combined to eventually yield the RBE for cell transformation. We show that the Local Effect Model is capable of predicting the RBE of neoplastic cell transformation for a broad range of ions and energies. The comparison of experimental data (4) with model calculations shows a reasonable agreement. We find that the cluster extension results in a better representation of the measured RBE values. With this model it should be possible to better

  20. Rapid 3D Refractive-Index Imaging of Live Cells in Suspension without Labeling Using Dielectrophoretic Cell Rotation.

    Science.gov (United States)

    Habaza, Mor; Kirschbaum, Michael; Guernth-Marschner, Christian; Dardikman, Gili; Barnea, Itay; Korenstein, Rafi; Duschl, Claus; Shaked, Natan T

    2017-02-01

    A major challenge in the field of optical imaging of live cells is achieving rapid, 3D, and noninvasive imaging of isolated cells without labeling. If successful, many clinical procedures involving analysis and sorting of cells drawn from body fluids, including blood, can be significantly improved. A new label-free tomographic interferometry approach is presented. This approach provides rapid capturing of the 3D refractive-index distribution of single cells in suspension. The cells flow in a microfluidic channel, are trapped, and then rapidly rotated by dielectrophoretic forces in a noninvasive and precise manner. Interferometric projections of the rotated cell are acquired and processed into the cellular 3D refractive-index map. Uniquely, this approach provides full (360°) coverage of the rotation angular range around any axis, and knowledge on the viewing angle. The experimental demonstrations presented include 3D, label-free imaging of cancer cells and three types of white blood cells. This approach is expected to be useful for label-free cell sorting, as well as for detection and monitoring of pathological conditions resulting in cellular morphology changes or occurrence of specific cell types in blood or other body fluids.

  1. Combination of Synthetic Chemistry and Live-Cell Imaging Identified a Rapid Cell Division Inhibitor in Tobacco and Arabidopsis thaliana.

    Science.gov (United States)

    Nambo, Masakazu; Kurihara, Daisuke; Yamada, Tomomi; Nishiwaki-Ohkawa, Taeko; Kadofusa, Naoya; Kimata, Yusuke; Kuwata, Keiko; Umeda, Masaaki; Ueda, Minako

    2016-11-01

    Cell proliferation is crucial to the growth of multicellular organisms, and thus the proper control of cell division is important to prevent developmental arrest or overgrowth. Nevertheless, tools for controlling cell proliferation are still poor in plant. To develop novel tools, we focused on a specific compound family, triarylmethanes, whose members show various antiproliferative activities in animals. By combining organic chemistry to create novel and diverse compounds containing the triarylmethyl moiety and biological screens based on live-cell imaging of a fluorescently labeled tobacco Bright Yellow-2 (BY-2) culture cell line (Nicotiana tabacum), we isolated (3-furyl)diphenylmethane as a strong but partially reversible inhibitor of plant cell division. We also found that this agent had efficient antiproliferative activity in developing organs of Arabidopsis thaliana without causing secondary defects in cell morphology, and induced rapid cell division arrest independent of the cell cycle stage. Given that (3-furyl)diphenylmethane did not affect the growth of a human cell line (HeLa) and a budding yeast (Saccharomyces cerevisiae), it should act specifically on plants. Taking our results together, we propose that the combination of desired chemical synthesis and detailed biological analysis is an effective tool to create novel drugs, and that (3-furyl)diphenylmethane is a specific antiproliferative agent for plants. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  2. The donor advocacy team: a risk management program for living organ, tissue, and cell transplant donors.

    Science.gov (United States)

    Eguchi, Susumu; Soyama, Akihiko; Nagai, Kazuhiro; Miyazaki, Yasushi; Kurihara, Shintaro; Hidaka, Masaaki; Ono, Shinichiro; Adachi, Tomohiko; Natsuda, Koji; Hara, Takanobu; Fujita, Fumihiko; Kanetaka, Kengo; Takatsuki, Mistuhisa

    2017-08-01

    Although the incidence of living donor death is low in Japan, statistics show one living liver donor death in more than 7000 living liver transplants. Thus, medical transplant personnel must recognize that the death of a living organ or tissue transplant donor can occur and develop an appropriate risk management program. We describe how Nagasaki University Hospital established and implemented a Donor Advocacy Team (DAT) program: a risk management program for initiation in the event of serious, persistent, or fatal impairment of an organ, tissue, or cell transplantation from a living donor. The purposes of the DAT program are as follows: 1. To disclose official information without delay. 2. To provide physical and psychological care to the patient experiencing impairment and their family. 3. To provide psychological care to the medical staff in charge of the transplant. 4. To standardize the responses of the diagnosis and treatment department staff and other hospital staff. 5. To minimize the damage that the whole medical transplantation system may suffer and leverage the occurrence for improvement. To address (1) and (5), actions, such as reporting and responses to the government, mass media, transplant-related societies, and organ transplant networks, have been established to ensure implementation.

  3. Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells.

    Science.gov (United States)

    Hedde, Per Niklas; Dörlich, René M; Blomley, Rosmarie; Gradl, Dietmar; Oppong, Emmanuel; Cato, Andrew C B; Nienhaus, G Ulrich

    2013-01-01

    Raster image correlation spectroscopy is a powerful tool to study fast molecular dynamics such as protein diffusion or receptor-ligand interactions inside living cells and tissues. By analysing spatio-temporal correlations of fluorescence intensity fluctuations from raster-scanned microscopy images, molecular motions can be revealed in a spatially resolved manner. Because of the diffraction-limited optical resolution, however, conventional raster image correlation spectroscopy can only distinguish larger regions of interest and requires low fluorophore concentrations in the nanomolar range. Here, to overcome these limitations, we combine raster image correlation spectroscopy with stimulated emission depletion microscopy. With imaging experiments on model membranes and live cells, we show that stimulated emission depletion-raster image correlation spectroscopy offers an enhanced multiplexing capability because of the enhanced spatial resolution as well as access to 10-100 times higher fluorophore concentrations.

  4. A genetically encoded multifunctional unnatural amino acid for versatile protein manipulations in living cells.

    Science.gov (United States)

    Ge, Yun; Fan, Xinyuan; Chen, Peng R

    2016-12-01

    The genetic code expansion strategy allowed incorporation of unnatural amino acids (UAAs) bearing diverse functional groups into proteins, providing a powerful toolkit for protein manipulation in living cells. We report a multifunctional UAA, N ε - p -azidobenzyloxycarbonyl lysine (PABK), that possesses a panel of unique properties capable of fulfilling various protein manipulation purposes. In addition to being used as a bioorthogonal ligation handle, an infrared probe and a photo-affinity reagent, PABK was shown to be chemically decaged by trans -cyclooctenols via a strain-promoted 1,3-dipolar cycloaddition, which provides a new bioorthogonal cleavage strategy for intracellular protein activation. The biocompatibility and efficiency of this method were demonstrated by decaging of a PABK-caged firefly luciferase under living conditions. We further extended this method to chemically rescue a bacterial toxin OspF inside mammalian host cells.

  5. Force probing of individual molecules inside the living cell is now a reality.

    Science.gov (United States)

    Oddershede, Lene B

    2012-11-01

    Biological systems can be quantitatively explored using single-molecule manipulation techniques such as optical or magnetic tweezers or atomic force microscopy. Though a plethora of discoveries have been accomplished using single-molecule manipulation techniques in vitro, such investigations constantly face the criticism that conditions are too far from being physiologically relevant. Technical achievements now allow scientists to take the next step: to use single-molecule manipulation techniques quantitatively in vivo. Considerable progress has been accomplished in this realm; for example, the interaction between a protein and the membrane of a living cell has been probed, the mechanical properties of individual proteins central for cellular adhesion have been measured and even the action of molecular motors in living cells has been quantified. Here, we review the progress of in vivo single-molecule manipulation with a focus on the special challenges posed by in vivo conditions and how these can be overcome.

  6. 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...

  7. Application of Live-Cell RNA Imaging Techniques to the Study of Retroviral RNA Trafficking

    Directory of Open Access Journals (Sweden)

    Darrin V. Bann

    2012-06-01

    Full Text Available Retroviruses produce full-length RNA that serves both as a genomic RNA (gRNA, which is encapsidated into virus particles, and as an mRNA, which directs the synthesis of viral structural proteins. However, we are only beginning to understand the cellular and viral factors that influence trafficking of retroviral RNA and the selection of the RNA for encapsidation or translation. Live cell imaging studies of retroviral RNA trafficking have provided important insight into many aspects of the retrovirus life cycle including transcription dynamics, nuclear export of viral RNA, translational regulation, membrane targeting, and condensation of the gRNA during virion assembly. Here, we review cutting-edge techniques to visualize single RNA molecules in live cells and discuss the application of these systems to studying retroviral RNA trafficking.

  8. 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.

  9. Transport of Ebolavirus Nucleocapsids Is Dependent on Actin Polymerization: Live-Cell Imaging Analysis of Ebolavirus-Infected Cells.

    Science.gov (United States)

    Schudt, Gordian; Dolnik, Olga; Kolesnikova, Larissa; Biedenkopf, Nadine; Herwig, Astrid; Becker, Stephan

    2015-10-01

    Transport of ebolavirus (EBOV) nucleocapsids from perinuclear viral inclusions, where they are formed, to the site of budding at the plasma membrane represents an obligatory step of virus assembly. Until now, no live-cell studies on EBOV nucleocapsid transport have been performed, and participation of host cellular factors in this process, as well as the trajectories and speed of nucleocapsid transport, remain unknown. Live-cell imaging of EBOV-infected cells treated with different inhibitors of cellular cytoskeleton was used for the identification of cellular proteins involved in the nucleocapsid transport. EBOV nucleocapsids were visualized by expression of green fluorescent protein (GFP)-labeled nucleocapsid viral protein 30 (VP30) in EBOV-infected cells. Incorporation of the fusion protein VP30-GFP into EBOV nucleocapsids was confirmed by Western blot and indirect immunofluorescence analyses. Importantly, VP30-GFP fluorescence was readily detectable in the densely packed nucleocapsids inside perinuclear viral inclusions and in the dispersed rod-like nucleocapsids located outside of viral inclusions. Live-cell imaging of EBOV-infected cells revealed exit of single nucleocapsids from the viral inclusions and their intricate transport within the cytoplasm before budding at the plasma membrane. Nucleocapsid transport was arrested upon depolymerization of actin filaments (F-actin) and inhibition of the actin-nucleating Arp2/3 complex, and it was not altered upon depolymerization of microtubules or inhibition of N-WASP. Actin comet tails were often detected at the rear end of nucleocapsids. Marginally located nucleocapsids entered filopodia, moved inside, and budded from the tip of these thin cellular protrusions. Live-cell imaging of EBOV-infected cells revealed actin-dependent long-distance transport of EBOV nucleocapsids before budding at the cell surface. These findings provide useful insights into EBOV assembly and have potential application in the development

  10. Correlating cell architecture with osteogenesis: first steps towards live single cell monitoring

    OpenAIRE

    A-K Born; M Rottmar; S Lischer; M Pleskova; A Bruinink; K Maniura-Weber

    2009-01-01

    Cell shape and regulation of biological processes such as proliferation and differentiation are to a large degree connected. Investigation of the possible relationship between cell shape and function is therefore important for developing new material concepts for medical applications as well as developing novel cell based sensors. Cell spreading requires a firm contact with the underlying substrate, with focal contacts (FC) being the primary sites of adhesion. They consist of a large number o...

  11. Live-cell super-resolution imaging of intrinsically fast moving flagellates

    Science.gov (United States)

    Glogger, M.; Stichler, S.; Subota, I.; Bertlein, S.; Spindler, M.-C.; Teßmar, J.; Groll, J.; Engstler, M.; Fenz, S. F.

    2017-02-01

    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 μs. 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. , which features invited work from the best early-career researchers working within the scope of J Phys D. This project is part of the Journal of Physics series’ 50th anniversary celebrations in 2017. Susanne Fenz was selected by the Editorial Board of J Phys D as an Emerging Talent/Leader.

  12. The Role of the WI-38 Cell Strain in Saving Lives and Reducing Morbidity

    OpenAIRE

    S. J. Olshansky; L. Hayflick

    2017-01-01

    The modern success story of vaccinations involves a historical chain of events that transformed the discovery that vaccines worked, to administering them to the population. We estimate the number of lives saved and morbidity reduction associated with the discovery of the first human cell strain used for the production of licensed human virus vaccines, known as WI-38. The diseases studied include poliomyelitis, measles, mumps, rubella, varicella (chicken pox), herpes zoster, adenovirus, rabies...

  13. A Fluorogenic TMP-tag for High Signal-to-Background Intracellular Live Cell Imaging

    OpenAIRE

    Jing, Chaoran; Cornish, Virginia W.

    2013-01-01

    Developed to compliment the use of fluorescent proteins in live cell imaging, chemical tags enjoy the benefit of modular incorporation of organic fluorophores, opening the possibility of high photon output and special photophysical properties. However, the theoretical challenge in using chemical tags as opposed to fluorescent proteins for high-resolution imaging is background noise from unbound and/or non-specifically bound ligand-fluorophore. We envisioned we could overcome this limit by eng...

  14. The radiation effects on the living cell; Les effets des rayonnements sur la cellule vivante

    Energy Technology Data Exchange (ETDEWEB)

    Sage, E.; Dutrillaux, B.; Soussi, Th. [Institut Curie, 75 - Paris (France)]|[Centre National de la Recherche Scientifique (CNRS), 75 - Paris (France); Boiteux, S.; Lopez, B. [CEA-CNRS/Fontenay-aux-Roses, 92 (France); Feunteun, J. [Institut Gustave Roussy, 94 - Villejuif (France)

    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.)

  15. A coumarin-quinolinium-based fluorescent probe for ratiometric sensing of sulfite in living cells.

    Science.gov (United States)

    Tan, Li; Lin, Weiying; Zhu, Sasa; Yuan, Lin; Zheng, Kaibo

    2014-07-14

    Based on a novel coumarin-quinolinium platform, probe 2 was rationally designed and synthesized as a novel ratiometric fluorescent sensor for sulfite anions. The probe exhibited a wide dynamic concentration range for sulfite anions in a PBS buffer (containing 1 mg mL(-1) BSA). More importantly, the probe was suitable for ratiometric fluorescence imaging in living cells with high sensitivity, favorable selectivity, and minimal cytotoxicity.

  16. Non-invasive single-cell biomechanical analysis using live-imaging datasets.

    Science.gov (United States)

    Pearson, Yanthe E; Lund, Amanda W; Lin, Alex W H; Ng, Chee P; Alsuwaidi, Aysha; Azzeh, Sara; Gater, Deborah L; Teo, Jeremy C M

    2016-09-01

    The physiological state of a cell is governed by a multitude of processes and can be described by a combination of mechanical, spatial and temporal properties. Quantifying cell dynamics at multiple scales is essential for comprehensive studies of cellular function, and remains a challenge for traditional end-point assays. We introduce an efficient, non-invasive computational tool that takes time-lapse images as input to automatically detect, segment and analyze unlabeled live cells; the program then outputs kinematic cellular shape and migration parameters, while simultaneously measuring cellular stiffness and viscosity. We demonstrate the capabilities of the program by testing it on human mesenchymal stem cells (huMSCs) induced to differentiate towards the osteoblastic (huOB) lineage, and T-lymphocyte cells (T cells) of naïve and stimulated phenotypes. The program detected relative cellular stiffness differences in huMSCs and huOBs that were comparable to those obtained with studies that utilize atomic force microscopy; it further distinguished naïve from stimulated T cells, based on characteristics necessary to invoke an immune response. In summary, we introduce an integrated tool to decipher spatiotemporal and intracellular dynamics of cells, providing a new and alternative approach for cell characterization. © 2016. Published by The Company of Biologists Ltd.

  17. The magnetic introduction of magnetite nanoparticles into live cells for radiosensibility enhancement

    Science.gov (United States)

    Yurenya, Anton Y.; Polikarpov, Mikhail A.; Chukalova, Aynur A.; Moskaleva, Elizaveta Y.; Taldenkov, Alexander N.; Panchenko, Vladislav Y.

    2017-04-01

    Earlier we proposed a new radiotherapy enhancement method that entails the administration of 57Fe iron-oxide nanoparticles into the cells [5]. 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 2·105 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 107 particles/cell. Most probably much of these particles penetrated into cells.

  18. Magnetic nanoparticle clusters radiosensitise human nasopharyngeal and lung cancer cells after alternating magnetic field treatment.

    Science.gov (United States)

    Ma, Jia; Zhang, Zhiping; Zhang, Zhanjie; Huang, Jing; Qin, You; Li, Xu; Liu, Hongli; Yang, Kunyu; Wu, Gang

    2015-01-01

    Heat generated by magnetic nanoparticle clusters (MNCs) in an alternating magnetic field (AMF) can be used for hyperthermia cancer treatment. Here, we have synthesised polyacrylic acid-coated MNCs according to previous report, with the ability to increase particle stability in suspension. Radiosensitisation effects of the MNCs under an AMF were investigated in vitro and in vivo. MTT assay, flow cytometry, clone formation assay, Western blotting, and a γ-H2AX experiment were used to explore the biocompatibility and radiosensitisation effect of the MNCs and their putative radiosensitisation mechanism. An NCI-H460 mouse xenograft model was used to investigate the anti-tumour effect under an AMF in vivo. The temperature of MNC fluids at different concentrations (200 μg/mL to 2 mg/mL) increased rapidly. The MNCs were endocytosed by the cells and were found to be biocompatible. Hsp70 and caspase-3 were found to be up-regulated upon MNCs under an AMF, radiation, and combination of both treatments. MNCs under an AMF efficiently radiosensitised both CNE-2 cells and NCI-H460 cells. Finally, the tumour inhibition rate after treatment with MNCs under an AMF and radiation was significantly higher than that after either treatment alone. The mechanism of radiosensitisation putatively involves inhibition of DNA repair and induction of apoptosis. The MNC fluids under an AMF enhanced the radiosensitivity of tumour cells both in vitro and in vivo.

  19. A Fluid Membrane-Based Soluble Ligand Display System for Live CellAssays

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Jwa-Min; Nair, Pradeep N.; Neve, Richard M.; Gray, Joe W.; Groves, Jay T.

    2005-10-14

    Cell communication modulates numerous biological processes including proliferation, apoptosis, motility, invasion and differentiation. Correspondingly, there has been significant interest in the development of surface display strategies for the presentation of signaling molecules to living cells. This effort has primarily focused on naturally surface-bound ligands, such as extracellular matrix components and cell membranes. Soluble ligands (e.g. growth factors and cytokines) play an important role in intercellular communications, and their display in a surface-bound format would be of great utility in the design of array-based live cell assays. Recently, several cell microarray systems that display cDNA, RNAi, or small molecules in a surface array format were proven to be useful in accelerating high-throughput functional genetic studies and screening therapeutic agents. These surface display methods provide a flexible platform for the systematic, combinatorial investigation of genes and small molecules affecting cellular processes and phenotypes of interest. In an analogous sense, it would be an important advance if one could display soluble signaling ligands in a surface assay format that allows for systematic, patterned presentation of soluble ligands to live cells. Such a technique would make it possible to examine cellular phenotypes of interest in a parallel format with soluble signaling ligands as one of the display parameters. Herein we report a ligand-modified fluid supported lipid bilayer (SLB) assay system that can be used to functionally display soluble ligands to cells in situ (Figure 1A). By displaying soluble ligands on a SLB surface, both solution behavior (the ability to become locally enriched by reaction-diffusion processes) and solid behavior (the ability to control the spatial location of the ligands in an open system) could be combined. The method reported herein benefits from the naturally fluid state of the supported membrane, which allows

  20. Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells.

    Science.gov (United States)

    Rizzuto, R; Brini, M; Pizzo, P; Murgia, M; Pozzan, T

    1995-06-01

    It has recently been demonstrated that the green fluorescent protein (GFP) of the jellyfish Aequorea victoria retains its fluorescent properties when recombinantly expressed in both prokaryotic (Escherichia coli) and eukaryotic (Caenorhabditis elegans and Drosophila melanogaster) living cells; it can therefore be used as a powerful marker of gene expression in vivo. The specific targeting of recombinant GFP within cells would allow it to be used for even more applications, but no information is yet available on the possibility of targeting GFP to intracellular organelles. In this study, we show that the GFP cDNA can be expressed at high levels in cultured mammalian cells; the recombinant polypeptide is highly fluorescent and is exclusively localized in the cytosol. Furthermore, we have modified the GFP cDNA to include a mitochondrial targeting sequence (and a strong immunological epitope at the amino terminus of the encoded polypeptide). When transiently transfected into mammalian cells, this construct drives the expression of a strongly fluorescent GFP chimera which selectively localizes to the mitochondria. We also describe two of the many possible applications of this recombinant GFP in physiological studies. The targeted chimera allows the visualization of mitochondrial movement in living cells. Also, unlike dyes such as rhodamine, it reveals morphological changes induced in mitochondria by drugs that collapse the organelle membrane potential. Moreover, when GFP is cotransfected with a membrane receptor, such as the alpha 1-adrenergic receptor, the fluorescence of the GFP in intact