WorldWideScience

Sample records for intermittent live cell

  1. Intermittent Fluorescence Oscillations in Lipid Droplets in a Live Normal and Lung Cancer Cell: Time-Resolved Confocal Microscopy.

    Science.gov (United States)

    Chowdhury, Rajdeep; Amin, Md Asif; Bhattacharyya, Kankan

    2015-08-27

    Intermittent structural oscillation in the lipid droplets of live lung cells is monitored using time-resolved confocal microscopy. Significant differences are observed between the lung cancer cell (A549) and normal (nonmalignant) lung cell (WI38). For this study, the lipid droplets are covalently labeled with a fluorescent dye, coumarin maleimide (7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin, CPM). The number of lipid droplets in the cancer cell is found to be ∼20-fold higher than that in the normal (nonmalignant) cell. The fluctuation in the fluorescence intensity of the dye (CPM) is attributed to the red-ox processes and periodic formation/rupture of the S-CPM bond. The amount of reactive oxygen species (ROS) is much higher in a cancer cell. This is manifested in faster oscillations (0.9 ± 0.3 s) in cancer cells compared to that in the normal cells (2.8 ± 0.7 s). Solvation dynamics in the lipid droplets of cancer cells is slower compared to that in the normal cell.

  2. Immobilization method of yeast cells for intermittent contact mode imaging using the atomic force microscope

    International Nuclear Information System (INIS)

    De, Tathagata; Chettoor, Antony M.; Agarwal, Pranav; Salapaka, Murti V.; Nettikadan, Saju

    2010-01-01

    The atomic force microscope (AFM) is widely used for studying the surface morphology and growth of live cells. There are relatively fewer reports on the AFM imaging of yeast cells (Kasas and Ikai, 1995), (Gad and Ikai, 1995). Yeasts have thick and mechanically strong cell walls and are therefore difficult to attach to a solid substrate. In this report, a new immobilization technique for the height mode imaging of living yeast cells in solid media using AFM is presented. The proposed technique allows the cell surface to be almost completely exposed to the environment and studied using AFM. Apart from the new immobilization protocol, for the first time, height mode imaging of live yeast cell surface in intermittent contact mode is presented in this report. Stable and reproducible imaging over a 10-h time span is observed. A significant improvement in operational stability will facilitate the investigation of growth patterns and surface patterns of yeast cells.

  3. Live-cell imaging.

    Science.gov (United States)

    Cole, Richard

    2014-01-01

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

  4. Microencapsulation Of Living Cells

    Science.gov (United States)

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

    1989-01-01

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

  5. Red cell survival and sequestration in acute intermittent porphyria

    International Nuclear Information System (INIS)

    Nawalkha, P.L.; Soni, S.G.; Agrawal, V.K.; Misra, S.N.

    1980-01-01

    Life span and sequestration of red cells have been studied in twenty one proved cases of acute intermittent porphyria of different age and sex group from Bikaner District, Rajasthan State (India). Chromium-51 labelled red cells were used in the study and the excess count method of Bughe Jones and Szur was used to calculate the index of sequestration. The mean apparent half survival time of erythrocytes in the control subjects was 25.9 +- 2.9 (S.D.) days and the same in the prophyria patients was 27.0 +- 3.8 days. This shows that the life span of red cells is normal in both the patient and the control. Excess destruction of red blood cells was found to take place in either spleen or liver in the disease and no excess accumulation of erythrocytes occurred over spleen as compared to liver. (M.G.B.)

  6. Physiological Adaptations to Hypoxic vs. Normoxic Training during Intermittent Living High

    Directory of Open Access Journals (Sweden)

    Stefan De Smet

    2017-05-01

    Full Text Available In the setting of “living high,” it is unclear whether high-intensity interval training (HIIT should be performed “low” or “high” to stimulate muscular and performance adaptations. Therefore, 10 physically active males participated in a 5-week “live high-train low or high” program (TR, whilst eight subjects were not engaged in any altitude or training intervention (CON. Five days per week (~15.5 h per day, TR was exposed to normobaric hypoxia simulating progressively increasing altitude of ~2,000–3,250 m. Three times per week, TR performed HIIT, administered as unilateral knee-extension training, with one leg in normobaric hypoxia (~4,300 m; TRHYP and with the other leg in normoxia (TRNOR. “Living high” elicited a consistent elevation in serum erythropoietin concentrations which adequately predicted the increase in hemoglobin mass (r = 0.78, P < 0.05; TR: +2.6%, P < 0.05; CON: −0.7%, P > 0.05. Muscle oxygenation during training was lower in TRHYP vs. TRNOR (P < 0.05. Muscle homogenate buffering capacity and pH-regulating protein abundance were similar between pretest and posttest. Oscillations in muscle blood volume during repeated sprints, as estimated by oscillations in NIRS-derived tHb, increased from pretest to posttest in TRHYP (~80%, P < 0.01 but not in TRNOR (~50%, P = 0.08. Muscle capillarity (~15% as well as repeated-sprint ability (~8% and 3-min maximal performance (~10–15% increased similarly in both legs (P < 0.05. Maximal isometric strength increased in TRHYP (~8%, P < 0.05 but not in TRNOR (~4%, P > 0.05. In conclusion, muscular and performance adaptations were largely similar following normoxic vs. hypoxic HIIT. However, hypoxic HIIT stimulated adaptations in isometric strength and muscle perfusion during intermittent sprinting.

  7. Stream biofilm responses to flow intermittency: from cells to ecosystems

    Directory of Open Access Journals (Sweden)

    Sergi eSabater

    2016-03-01

    Full Text Available Temporary streams are characterized by the alternation of dry and wet hydrological phases, creating both a harsh environment for the biota as well as a high diversity of opportunities for adaptation. These systems are eminently microbial-based during several of these hydrological phases, and those growing on all solid substrata (biofilms accordingly change their physical structure and community composition. Biofilms experience large decreases on cell densities and biomass, both of bacteria and algae, during dryness. Algal and bacterial communities show remarkable decreases in their diversity, at least locally (at the habitat scale. Biofilms also respond with significant physiological plasticity to each of the hydrological changes. The decreasing humidity of the substrata through the drying process, and the changing quantity and quality of organic matter and nutrients available in the stream during that process, causes unequal responses on the biofilm bacteria and algae. Biofilm algae are affected faster than bacteria by the hydric stress, and as a result the ecosystem respiration resists longer than gross primary production to the increasing duration of flow intermittency. This response implies enhancing ecosystem heterotrophy, a pattern that can be exacerbated in temporary streams suffering of longer dry periods under global change.

  8. Dynamic water management of polymer electrolyte membrane fuel cells using intermittent RH control

    KAUST Repository

    Hussaini, I.S.; Wang, C.Y.

    2010-01-01

    A novel method of water management of polymer electrolyte membrane (PEM) fuel cells using intermittent humidification is presented in this study. The goal is to maintain the membrane close to full humidification, while eliminating channel flooding

  9. COMPARISON OF LIVE HIGH: TRAIN LOW ALTITUDE AND INTERMITTENT HYPOXIC EXPOSURE

    Directory of Open Access Journals (Sweden)

    Clare E. Humberstone-Gough

    2013-09-01

    Full Text Available Live High:Train Low (LHTL altitude training is a popular ergogenic aid amongst athletes. An alternative hypoxia protocol, acute (60-90 min daily Intermittent Hypoxic Exposure (IHE, has shown potential for improving athletic performance. The aim of this study was to compare directly the effects of LHTL and IHE on the running and blood characteristics of elite triathletes. Changes in total haemoglobin mass (Hbmass, maximal oxygen consumption (VO2max, velocity at VO2max (vVO2max, time to exhaustion (TTE, running economy, maximal blood lactate concentration ([La] and 3 mM [La] running speed were compared following 17 days of LHTL (240 h of hypoxia, IHE (10.2 h of hypoxia or Placebo treatment in 24 Australian National Team triathletes (7 female, 17 male. There was a clear 3.2 ± 4.8% (mean ± 90% confidence limits increase in Hbmass following LHTL compared with Placebo, whereas the corresponding change of -1.4 ± 4.5% in IHE was unclear. Following LHTL, running economy was 2.8 ± 4.4% improved compared to IHE and 3mM [La] running speed was 4.4 ± 4.5% improved compared to Placebo. After IHE, there were no beneficial changes in running economy or 3mM [La] running speed compared to Placebo. There were no clear changes in VO2max, vVO2max and TTE following either method of hypoxia. The clear difference in Hbmass response between LHTL and IHE indicated that the dose of hypoxia in IHE was insufficient to induce accelerated erythropoiesis. Improved running economy and 3mM [La] running speed following LHTL suggested that this method of hypoxic exposure may enhance performance at submaximal running speeds. Overall, there was no evidence to support the use of IHE in elite triathletes

  10. Intermittent Hypoxia Alters Gene Expression in Peripheral Blood Mononuclear Cells of Healthy Volunteers.

    Science.gov (United States)

    Polotsky, Vsevolod Y; Bevans-Fonti, Shannon; Grigoryev, Dmitry N; Punjabi, Naresh M

    2015-01-01

    Obstructive sleep apnea is associated with high cardiovascular morbidity and mortality. Intermittent hypoxia of obstructive sleep apnea is implicated in the development and progression of insulin resistance and atherosclerosis, which have been attributed to systemic inflammation. Intermittent hypoxia leads to pro-inflammatory gene up-regulation in cell culture, but the effects of intermittent hypoxia on gene expression in humans have not been elucidated. A cross-over study was performed exposing eight healthy men to intermittent hypoxia or control conditions for five hours with peripheral blood mononuclear cell isolation before and after exposures. Total RNA was isolated followed by gene microarrays and confirmatory real time reverse transcriptase PCR. Intermittent hypoxia led to greater than two fold up-regulation of the pro-inflammatory gene toll receptor 2 (TLR2), which was not increased in the control exposure. We hypothesize that up-regulation of TLR2 by intermittent hypoxia may lead to systemic inflammation, insulin resistance and atherosclerosis in patients with obstructive sleep apnea.

  11. The Cytotoxic Role of Intermittent High Glucose on Apoptosis and Cell Viability in Pancreatic Beta Cells

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    2014-01-01

    Full Text Available Objectives. Glucose fluctuations are both strong predictor of diabetic complications and crucial factor for beta cell damages. Here we investigated the effect of intermittent high glucose (IHG on both cell apoptosis and proliferation activity in INS-1 cells and the potential mechanisms. Methods. Cells were treated with normal glucose (5.5 mmol/L, constant high glucose (CHG (25 mmol/L, and IHG (rotation per 24 h in 11.1 or 25 mmol/L for 7 days. Reactive oxygen species (ROS, xanthine oxidase (XOD level, apoptosis, cell viability, cell cycle, and expression of cyclinD1, p21, p27, and Skp2 were determined. Results. We found that IHG induced more significant apoptosis than CHG and normal glucose; intracellular ROS and XOD levels were more markedly increased in cells exposed to IHG. Cells treated with IHG showed significant decreased cell viability and increased cell proportion in G0/G1 phase. Cell cycle related proteins such as cyclinD1 and Skp2 were decreased significantly, but expressions of p27 and p21 were increased markedly. Conclusions. This study suggested that IHG plays a more toxic effect including both apoptosis-inducing and antiproliferative effects on INS-1 cells. Excessive activation of cellular stress and regulation of cyclins might be potential mechanism of impairment in INS-1 cells induced by IHG.

  12. Intermittent fasting preserves beta-cell mass in obesity-induced diabetes via the autophagy-lysosome pathway.

    Science.gov (United States)

    Liu, Haiyan; Javaheri, Ali; Godar, Rebecca J; Murphy, John; Ma, Xiucui; Rohatgi, Nidhi; Mahadevan, Jana; Hyrc, Krzysztof; Saftig, Paul; Marshall, Connie; McDaniel, Michael L; Remedi, Maria S; Razani, Babak; Urano, Fumihiko; Diwan, Abhinav

    2017-01-01

    Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.

  13. Stream biofilm responses to flow intermittency: from cells to ecosystems

    OpenAIRE

    Sergi eSabater; Sergi eSabater; Xisca eTimoner; Carles eBorrego; Carles eBorrego; Vicenç eAcuña

    2016-01-01

    Temporary streams are characterized by the alternation of dry and wet hydrological phases, creating both a harsh environment for the biota as well as a high diversity of opportunities for adaptation. These systems are eminently microbial-based during several of these hydrological phases, and those growing on all solid substrata (biofilms) accordingly change their physical structure and community composition. Biofilms experience large decreases on cell densities and biomass, both of bacteria a...

  14. Stream Biofilm Responses to Flow Intermittency: From Cells to Ecosystems

    OpenAIRE

    Sabater, Sergi; Timoner, Xisca; Borrego, Carles; Acuña, Vicenç

    2016-01-01

    Temporary streams are characterized by the alternation of dry and wet hydrological phases, creating both a harsh environment for the biota as well as a high diversity of opportunities for adaptation. These systems are mainly microbial-based during several of these hydrological phases, and those growing on all solid substrata (biofilms) accordingly change their physical structure and community composition. Biofilms experience large decreases in cell densities and biomass, both of bacteria and ...

  15. 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.

  16. Diffusion inside living human cells

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  17. Living and Training at 825 m for 8 Weeks Supplemented With Intermittent Hypoxic Training at 3,000 m Improves Blood Parameters and Running Performance.

    Science.gov (United States)

    Wonnabussapawich, Preetiwat; Hamlin, Michael J; Lizamore, Catherine A; Manimmanakorn, Nuttaset; Leelayuwat, Naruemon; Tunkamnerdthai, Orathai; Thuwakum, Worrawut; Manimmanakorn, Apiwan

    2017-12-01

    Wonnabussapawich, P, Hamlin, MJ, Lizamore, CA, Manimmanakorn, N, Leelayuwat, N, Tunkamnerdthai, O, Thuwakum, W, and Manimmanakorn, A. Living and training at 825 m for 8 weeks supplemented with intermittent hypoxic training at 3,000 m improves blood parameters and running performance. J Strength Cond Res 31(12): 3287-3294, 2017-We aimed to investigate the effect of an 8-week low-altitude training block supplemented with intermittent hypoxic training, on blood and performance parameters in soccer players. Forty university-level male soccer players were separated into altitude (n = 20, 825 m) or sea-level (n = 20, 125 m) groups. Before (1-2 days ago) and after (1 and 14 days later) training, players were asked to give a resting venous blood sample and complete a series of performance tests. Compared with sea level, the altitude group increased erythropoietin, red blood cell (RBC) count, and hematocrit 1 day after training (42.6 ± 24.0%, 1.8 ± 1.3%, 1.4 ± 1.1%, mean ± 95% confidence limits (CL), respectively). By 14 days after training, only RBC count and hemoglobin were substantially higher in the altitude compared with the sea-level group (3.2 ± 1.8%, 2.9 ± 2.1% respectively). Compared with sea level, the altitude group 1-2 days after training improved their 50-m (-2.9 ± 1.4%) and 2,800-m (-2.9 ± 4.4%) run times and demonstrated a higher maximal aerobic speed (4.7 ± 7.4%). These performance changes remained at 14 days after training with the addition of a likely higher estimated V[Combining Dot Above]O2max in the altitude compared with the sea-level group (3.2 ± 3.0%). Eight weeks of low-altitude training, supplemented with regular bouts of intermittent hypoxic training at higher altitude, produced beneficial performance improvements in team-sport athletes, which may increase the viability of such training to coaches and players that cannot access more traditional high altitude venues.

  18. 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.

  19. The effects of pentoxifylline on the survival of human glioma cells with continuous and intermittent stereotactic radiosurgery irradiation

    International Nuclear Information System (INIS)

    Eley, Kerry W.; Benedict, Stanley H.; Chung, Theodore D.K.; Kavanagh, Brian D.; Broaddus, William C.; Schmidt-Ullrich, Rupert K.A.; Lin, P.-S.

    2002-01-01

    Purpose: In linac-based stereotactic radiosurgery, treatment is delivered intermittently via multiple individual small radiotherapy arcs. The time lapses between the individual arcs permit greater damage repair and increased tumor cell survival in comparison with continuous irradiation. Because pentoxifylline (PTX) has been reported to prevent radiation-induced cell cycle arrest at the G2/M checkpoint, where damage repair is critically linked to cell survival, we hypothesized that PTX would exert a favorable radiosensitization effect by reducing the recovery observed during intermittent stereotactic radiosurgery. Methods and Materials: The human glioma cell line T98G was used to study the effects of continuous vs. intermittent irradiation with or without PTX. Cell cycle patterns were studied using flow cytometry. Clonogenic assays of single cells and spheroid outgrowth assays provided a quantitative measure of PTX-mediated radiosensitization. The PTX effect upon cells in low oxygen conditions was also studied in vitro after enzymatic oxygen scavenging. Results: Flow Cytometry: T98G cells exposed to both continuous and intermittent irradiation exhibit similar arrest at the G2/M checkpoint. The addition of 2 mM PTX significantly reduced the radiation-induced G2/M block in both irradiation schemes. Clonogenic Assays: The same PTX concentration applied before a continuous dose of 12 Gy, two intermittent doses of 6 Gy, or three intermittent doses of 4 Gy, all given within a 1-h interval, consistently caused radiosensitization. The drug enhancement ratios for PTX were 1.5, 2.7, and 6.0 for the continuous and two different intermittent dose schedules, respectively. Adding PTX after irradiation yielded lower enhancement ratios than pre-irradiation application. A similar pattern was observed after total doses of 4, 6, 9, or 12 Gy, as well. In low oxygen conditions, PTX was seen to have the same effects as in normoxic conditions. Spheroid Outgrowth Assays: The in vitro PTX

  20. Quantification of nanowire uptake by live cells

    KAUST Repository

    Margineanu, Michael B.

    2015-01-01

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

  1. Polyvalent Display of Biomolecules on Live Cells.

    Science.gov (United States)

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

    2018-06-04

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

  2. Dynamic water management of polymer electrolyte membrane fuel cells using intermittent RH control

    KAUST Repository

    Hussaini, I.S.

    2010-06-01

    A novel method of water management of polymer electrolyte membrane (PEM) fuel cells using intermittent humidification is presented in this study. The goal is to maintain the membrane close to full humidification, while eliminating channel flooding. The entire cycle is divided into four stages: saturation and de-saturation of the gas diffusion layer followed by de-hydration and hydration of membrane. By controlling the duration of dry and humid flows, it is shown that the cell voltage can be maintained within a narrow band. The technique is applied on experimental test cells using both plain and hydrophobic materials for the gas diffusion layer and an improvement in performance as compared to steady humidification is demonstrated. Duration of dry and humid flows is determined experimentally for several operating conditions. © 2010 Elsevier B.V. All rights reserved.

  3. Hybrid Microgrid Model based on Solar Photovoltaics with Batteries and Fuel Cells system for intermittent applications

    Science.gov (United States)

    Patterson, Maxx

    Microgrids are a subset of the modern power structure; using distributed generation (DG) to supply power to communities rather than vast regions. The reduced scale mitigates loss allowing the power produced to do more with better control, giving greater security, reliability, and design flexibility. This paper explores the performance and cost viability of a hybrid grid-tied microgrid that utilizes Photovoltaic (PV), batteries, and fuel cell (FC) technology. The concept proposes that each community home is equipped with more PV than is required for normal operation. As the homes are part of a microgrid, excess or unused energy from one home is collected for use elsewhere within the microgrid footprint. The surplus power that would have been discarded becomes a community asset, and is used to run intermittent services. In this paper, the modeled community does not have parking adjacent to each home allowing for the installment of a privately owned slower Level 2 charger, making EV ownership option untenable. A solution is to provide a Level 3 DC Quick Charger (DCQC) as the intermittent service. The addition of batteries and Fuel Cells are meant to increase load leveling, reliability, and instill limited island capability.

  4. Intermittent fasting during Ramadan attenuates proinflammatory cytokines and immune cells in healthy subjects.

    Science.gov (United States)

    Faris, Mo'ez Al-Islam E; Kacimi, Safia; Al-Kurd, Ref'at A; Fararjeh, Mohammad A; Bustanji, Yasser K; Mohammad, Mohammad K; Salem, Mohammad L

    2012-12-01

    Intermittent fasting and caloric restriction have been shown to extend life expectancy and reduce inflammation and cancer promotion in animal models. It was hypothesized that intermittent prolonged fasting practiced during the month of Ramadan (RIF) could positively affect the inflammatory state. To investigate this hypothesis, a cross-sectional study was designed to investigate the impact of RIF on selected inflammatory cytokines and immune biomarkers in healthy subjects. Fifty (21 men and 29 women) healthy volunteers who practiced Ramadan fasting were recruited for the investigation of circulating proinflammatory cytokines (interleukin [IL]-1β, IL-6, and tumor necrosis factor α), immune cells (total leukocytes, monocytes, granulocytes, and lymphocytes), and anthropometric and dietary assessments. The investigations were conducted 1 week before Ramadan fasting, at the end of the third week of Ramadan, and 1 month after the cessation of Ramadan month. The proinflammatory cytokines IL-1β, IL-6, and tumor necrosis factor α; systolic and diastolic blood pressures; body weight; and body fat percentage were significantly lower (P fasting. Immune cells significantly decreased during Ramadan but still remained within the reference ranges. These results indicate that RIF attenuates inflammatory status of the body by suppressing proinflammatory cytokine expression and decreasing body fat and circulating levels of leukocytes. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Intermittent Stem Cell Cycling Balances Self-Renewal and Senescence of the C. elegans Germ Line.

    Directory of Open Access Journals (Sweden)

    Amanda Cinquin

    2016-04-01

    Full Text Available Self-renewing organs often experience a decline in function in the course of aging. It is unclear whether chronological age or external factors control this decline, or whether it is driven by stem cell self-renewal-for example, because cycling cells exhaust their replicative capacity and become senescent. Here we assay the relationship between stem cell cycling and senescence in the Caenorhabditis elegans reproductive system, defining this senescence as the progressive decline in "reproductive capacity," i.e. in the number of progeny that can be produced until cessation of reproduction. We show that stem cell cycling diminishes remaining reproductive capacity, at least in part through the DNA damage response. Paradoxically, gonads kept under conditions that preclude reproduction keep cycling and producing cells that undergo apoptosis or are laid as unfertilized gametes, thus squandering reproductive capacity. We show that continued activity is in fact beneficial inasmuch as gonads that are active when reproduction is initiated have more sustained early progeny production. Intriguingly, continued cycling is intermittent-gonads switch between active and dormant states-and in all likelihood stochastic. Other organs face tradeoffs whereby stem cell cycling has the beneficial effect of providing freshly-differentiated cells and the detrimental effect of increasing the likelihood of cancer or senescence; stochastic stem cell cycling may allow for a subset of cells to preserve proliferative potential in old age, which may implement a strategy to deal with uncertainty as to the total amount of proliferation to be undergone over an organism's lifespan.

  6. 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.

  7. A Novel Chip for Cyclic Stretch and Intermittent Hypoxia Cell Exposures Mimicking Obstructive Sleep Apnea

    Directory of Open Access Journals (Sweden)

    Noelia Campillo

    2016-07-01

    Full Text Available Intermittent hypoxia (IH, a hallmark of obstructive sleep apnea (OSA, plays a critical role in the pathogenesis of OSA-associated morbidities, especially in the cardiovascular and respiratory systems. Oxidative stress and inflammation induced by IH are suggested as main contributors of end-organ dysfunction in OSA patients and animal models. Since the molecular mechanisms underlying these in vivo pathological responses remain poorly understood, implementation of experimental in vitro cell-based systems capable of inducing high-frequency IH would be highly desirable. Here, we describe the design, fabrication and validation of a versatile chip for subjecting cultured cells to fast changes in gas partial pressure and to cyclic stretch. The chip is fabricated with polydimethylsiloxane (PDMS and consists of a cylindrical well covered by a thin membrane. Cells cultured on top of the membrane can be subjected to fast changes in oxygen concentration (equilibrium time 6 s. Moreover, cells can be subjected to cyclic stretch at cardiac or respiratory frequencies independently or simultaneously. Rat bone marrow-derived mesenchymal stem cells (MSCs exposed to IH mimicking OSA and cyclic stretch at cardiac frequencies revealed that hypoxia-inducible factor 1α (HIF-1α expression was increased in response to both stimuli. Thus, the chip provides a versatile tool for the study of cellular responses to cyclical hypoxia and stretch.

  8. Circulating, cell-free DNA as a marker for exercise load in intermittent sports

    OpenAIRE

    Haller, Nils; Helmig, Susanne; Taenny, Pascal; Petry, Julian; Schmidt, Sebastian; Simon, Perikles

    2018-01-01

    Background Attempts to establish a biomarker reflecting individual player load in intermittent sports such as football have failed so far. Increases in circulating DNA (cfDNA) have been demonstrated in various endurance sports settings. While it has been proposed that cfDNA could be a suitable marker for player load in intermittent sports, the effects on cfDNA of repeated sprinting as an essential feature in intermittent sports are unknown. For the first time, we assessed both alterations of ...

  9. 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.

  10. Energy intermittency

    CERN Document Server

    Sorensen, Bent

    2014-01-01

    The first book to consider intermittency as a key point of an energy system, Energy Intermittency describes different levels of variability for traditional and renewable energy sources, presenting detailed solutions for handling energy intermittency through trade, collaboration, demand management, and active energy storage. Addressing energy supply intermittency systematically, this practical text:Analyzes typical time-distributions and intervals between episodes of demand-supply mismatch and explores their dependence on system layouts and energy source characteristicsSimulates scenarios regar

  11. Laser-Raman spectroscopy of living cells

    International Nuclear Information System (INIS)

    Webb, S.J.

    1980-01-01

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

  12. Intermittent Hypoxia Disrupts Glucose Homeostasis in Liver Cells in an Insulin-Dependent and Independent Manner

    Directory of Open Access Journals (Sweden)

    Chen Juan Gu

    2018-05-01

    Full Text Available Background/Aims: Obstructive sleep apnea is associated with diabetes and insulin resistance, but the underlying mechanisms remain unclear. The purpose of the current study was to determine the molecular effects of intermittent hypoxia (IH on hepatic insulin signaling and glucose homeostasis, and whether c-Jun NH2-terminal-kinase (JNK contributed to metabolic responses to IH in liver cells. Methods: The human HepG2 cells and rat FAO cells were exposed to 10, 30, 120, 240 or 360 cycles of IH (1% O2 for 60 s followed by 21% O2 for 60s, 7.5 cycles per hour or normoxia as a control. In a subgroup, we exposed cells to 360 cycles of IH with the JNK inhibitor SP600125. After IH exposure, cell glycogen content and glucose output were measured using colorimetric assay kits. Canonical insulin signaling and gluconeogenic genes were measured by western blot and quantitative polymerase chain reaction. Results: IH decreased insulin-stimulated protein kinase B (AKT/glycogen synthase kinase-3β (GSK-3β phosphorylation in a time-dependent manner, while inhibiting forkhead box protein O1 (FOXO1 expression and phosphoenolpyruvate carboxykinase (PEPCK transcription independent of insulin signaling. JNK inhibitor SP600125 partially restored AKT/ GSK-3β phosphorylation and glycogen synthesis, but did not affect other IH-induced glucose metabolic changes. Conclusion: IH in vitro impaired insulin signal transduction in liver cells as assessed by inhibited AKT/GSK-3β phosphorylation via JNK activation. IH inhibited FOXO1 and gluconeogenesis in an insulin-independent manner.

  13. Biomimetic silica encapsultation of living cells

    Science.gov (United States)

    Jaroch, David Benjamin

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

  14. 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.)

  15. Living labeling techniques of mesenchymal stem cells

    International Nuclear Information System (INIS)

    Dong Qingyu; Chen Li

    2007-01-01

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

  16. Circumventing photodamage in live-cell microscopy

    Science.gov (United States)

    Magidson, Valentin; Khodjakov, Alexey

    2013-01-01

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

  17. 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.

  18. Synthetic analog computation in living cells.

    Science.gov (United States)

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

    2013-05-30

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

  19. 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.

  20. On strain and stress in living cells

    Science.gov (United States)

    Cox, Brian N.; Smith, David W.

    2014-11-01

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

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

    Science.gov (United States)

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

    2012-06-07

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

  2. 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.

  3. 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

  4. Intermittent feeding as a factor enhancing hemopoietic stem cell proliferation and spleen colony formation in irradiated mice

    International Nuclear Information System (INIS)

    Kozubik, A.; Pospisil, M.

    1985-01-01

    The influence of metabolic stimulation induced by a 3 weeks' adaption of the animals to intermittent food intake on hemopoietic stem cells was investigated in mice. The methods used included transplantation of bone marrow to lethally irradiated recipients, assay of CFUs number, seeding efficiency, and incorporation of 125 iodode oxyuridine into the DNA of spleen cells. A stimulatory effect of the metabolically influenced hemopoietic environment on the proliferative activity in stem cell compartments and on the recovery of hemopoietic organs was demonstrated. These stimulatory effects were most marked when the bone marrow of metabolically influenced donors was transplanted to similarly influenced recipients. (orig.)

  5. Total intermittent Pringle maneuver during liver resection can induce intestinal epithelial cell damage and endotoxemia.

    Directory of Open Access Journals (Sweden)

    Simon A W G Dello

    Full Text Available OBJECTIVES: The intermittent Pringle maneuver (IPM is frequently applied to minimize blood loss during liver transection. Clamping the hepatoduodenal ligament blocks the hepatic inflow, which leads to a non circulating (hepatosplanchnic outflow. Also, IPM blocks the mesenteric venous drainage (as well as the splenic drainage with raising pressure in the microvascular network of the intestinal structures. It is unknown whether the IPM is harmful to the gut. The aim was to investigate intestinal epithelial cell damage reflected by circulating intestinal fatty acid binding protein levels (I-FABP in patients undergoing liver resection with IPM. METHODS: Patients who underwent liver surgery received total IPM (total-IPM or selective IPM (sel-IPM. A selective IPM was performed by selectively clamping the right portal pedicle. Patients without IPM served as controls (no-IPM. Arterial blood samples were taken immediately after incision, ischemia and reperfusion of the liver, transection, 8 hours after start of surgery and on the first post-operative day. RESULTS: 24 patients (13 males were included. 7 patients received cycles of 15 minutes and 5 patients received cycles of 30 minutes of hepatic inflow occlusion. 6 patients received cycles of 15 minutes selective hepatic occlusion and 6 patients underwent surgery without inflow occlusion. Application of total-IPM resulted in a significant increase in I-FABP 8 hours after start of surgery compared to baseline (p<0.005. In the no-IPM group and sel-IPM group no significant increase in I-FABP at any time point compared to baseline was observed. CONCLUSION: Total-IPM in patients undergoing liver resection is associated with a substantial increase in arterial I-FABP, pointing to intestinal epithelial injury during liver surgery. TRIAL REGISTRATION: ClinicalTrials.gov NCT01099475.

  6. Intermittent Hypoxia Affects the Spontaneous Differentiation In Vitro of Human Neutrophils into Long-Lived Giant Phagocytes

    Directory of Open Access Journals (Sweden)

    Larissa Dyugovskaya

    2016-01-01

    Full Text Available Previously we identified, for the first time, a new small-size subset of neutrophil-derived giant phagocytes (Gϕ which spontaneously develop in vitro without additional growth factors or cytokines. Gϕ are CD66b+/CD63+/MPO+/LC3B+ and are characterized by extended lifespan, large phagolysosomes, active phagocytosis, and reactive oxygen species (ROS production, and autophagy largely controls their formation. Hypoxia, and particularly hypoxia/reoxygenation, is a prominent feature of many pathological processes. Herein we investigated Gϕ formation by applying various hypoxic conditions. Chronic intermittent hypoxia (IH (29 cycles/day for 5 days completely abolished Gϕ formation, while acute IH had dose-dependent effects. Exposure to 24 h (56 IH cycles decreased their size, yield, phagocytic ability, autophagy, mitophagy, and gp91-phox/p22-phox expression, whereas under 24 h sustained hypoxia (SH the size and expression of LC3B and gp91-phox/p22-phox resembled Gϕ formed in normoxia. Diphenyl iodide (DPI, a NADPH oxidase inhibitor, as well as the PI3K/Akt and autophagy inhibitor LY294002 abolished Gϕ formation at all oxygen conditions. However, the potent antioxidant, N-acetylcysteine (NAC abrogated the effects of IH by inducing large CD66b+/LC3B+ Gϕ and increased both NADPH oxidase expression and phagocytosis. These findings suggest that NADPH oxidase, autophagy, and the PI3K/Akt pathway are involved in Gϕ development.

  7. Kinase Activity Studied in Living Cells Using an Immunoassay

    Science.gov (United States)

    Bavec, Aljos?a

    2014-01-01

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

  8. NFAT regulation of cystathionine γ-lyase expression in endothelial cells is impaired in rats exposed to intermittent hypoxia.

    Science.gov (United States)

    Gonzalez Bosc, Laura V; Osmond, Jessica M; Giermakowska, Wieslawa K; Pace, Carolyn E; Riggs, Jennifer L; Jackson-Weaver, Olan; Kanagy, Nancy L

    2017-04-01

    Sleep apnea is a risk factor for cardiovascular disease, and intermittent hypoxia (IH, 20 episodes/h of 5% O 2 -5% CO 2 for 7 h/day) to mimic sleep apnea increases blood pressure and impairs hydrogen sulfide (H 2 S)-induced vasodilation in rats. The enzyme that produces H 2 S, cystathionine γ-lyase (CSE), is decreased in rat mesenteric artery endothelial cells (EC) following in vivo IH exposure. In silico analysis identified putative nuclear factor of activated T cell (NFAT) binding sites in the CSE promoter. Therefore, we hypothesized that IH exposure reduces Ca 2+ concentration ([Ca 2+ ]) activation of calcineurin/NFAT to lower CSE expression and impair vasodilation. In cultured rat aortic EC, inhibiting calcineurin with cyclosporine A reduced CSE mRNA, CSE protein, and luciferase activity driven by a full-length but not a truncated CSE promoter. In male rats exposed to sham or IH conditions for 2 wk, [Ca 2+ ] in EC in small mesenteric arteries from IH rats was lower than in EC from sham rat arteries (Δfura 2 ratio of fluorescence at 340 to 380 nm from Ca 2+ free: IH = 0.05 ± 0.02, sham = 0.17 ± 0.03, P intermittent hypoxia to mimic sleep apnea, nuclear factor of activated T cells c3 nuclear translocation and CSE expression are decreased, concomitant with decreased CSE-dependent vasodilation. Copyright © 2017 the American Physiological Society.

  9. 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

  10. 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.

  11. An innovative intermittent hypoxia model for cell cultures allowing fast Po2 oscillations with minimal gas consumption.

    Science.gov (United States)

    Minoves, Mélanie; Morand, Jessica; Perriot, Frédéric; Chatard, Morgane; Gonthier, Brigitte; Lemarié, Emeline; Menut, Jean-Baptiste; Polak, Jan; Pépin, Jean-Louis; Godin-Ribuot, Diane; Briançon-Marjollet, Anne

    2017-10-01

    Performing hypoxia-reoxygenation cycles in cell culture with a cycle duration accurately reflecting what occurs in obstructive sleep apnea (OSA) patients is a difficult but crucial technical challenge. Our goal was to develop a novel device to expose multiple cell culture dishes to intermittent hypoxia (IH) cycles relevant to OSA with limited gas consumption. With gas flows as low as 200 ml/min, our combination of plate holders with gas-permeable cultureware generates rapid normoxia-hypoxia cycles. Cycles alternating 1 min at 20% O 2 followed by 1 min at 2% O 2 resulted in Po 2 values ranging from 124 to 44 mmHg. Extending hypoxic and normoxic phases to 10 min allowed Po 2 variations from 120 to 25 mmHg. The volume of culture medium or the presence of cells only modestly affected the Po 2 variations. In contrast, the nadir of the hypoxia phase increased when measured at different heights above the membrane. We validated the physiological relevance of this model by showing that hypoxia inducible factor-1α expression was significantly increased by IH exposure in human aortic endothelial cells, murine breast carcinoma (4T1) cells as well as in a blood-brain barrier model (2.5-, 1.5-, and 6-fold increases, respectively). In conclusion, we have established a new device to perform rapid intermittent hypoxia cycles in cell cultures, with minimal gas consumption and the possibility to expose several culture dishes simultaneously. This device will allow functional studies of the consequences of IH and deciphering of the molecular biology of IH at the cellular level using oxygen cycles that are clinically relevant to OSA. Copyright © 2017 the American Physiological Society.

  12. Long-term activity of covalent grafted biocatalysts during intermittent use of a glucose/O2 biofuel cell

    International Nuclear Information System (INIS)

    Merle, G.; Habrioux, A.; Servat, K.; Rolland, M.; Innocent, C.; Kokoh, K.B.; Tingry, S.

    2009-01-01

    The operational stability of enzymes in a concentric glucose/O 2 biofuel cell has been significantly improved with the synthesis of grafted enzyme electrodes compared to entrapped enzyme electrodes. The concentric device combined glucose electro-oxidation by glucose oxidase at the anode and oxygen electro-reduction by bilirubin oxidase at the cathode. The entrapped enzyme electrodes were prepared from physical immobilization of the enzymes by a polypyrrole polymer onto the electrode surface. The grafted enzyme electrodes were synthesized by grafting the enzymes via alkyl spacer arms to a poly(aminopropylpyrrole) film onto the electrode surface. From spectrophotometric and electrochemical analyses, it was demonstrated that the spacer arms increased the operational stability and enzyme mobility that favoured electron transfer from their active sites to the electrode. The maximum power output of the assembled biofuel cell was 20 μW cm -2 , at 0.20 V with 10 mM glucose in phosphate buffer pH 7.4. The grafted enzyme electrodes presented an unprecedented operational stability as the maximum of power density of the BFC remains constant after intermittent use over a 45-day period. This was a remarkable improvement compared to electrodes with entrapped enzymes, which lost 74% of their initial power density after intermittent use over a 17-day period

  13. Functional living biointerfaces to direct cell-material interaction

    OpenAIRE

    Rodrigo Navarro, Aleixandre

    2016-01-01

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

  14. Circulating, cell-free DNA as a marker for exercise load in intermittent sports.

    Science.gov (United States)

    Haller, Nils; Helmig, Susanne; Taenny, Pascal; Petry, Julian; Schmidt, Sebastian; Simon, Perikles

    2018-01-01

    Attempts to establish a biomarker reflecting individual player load in intermittent sports such as football have failed so far. Increases in circulating DNA (cfDNA) have been demonstrated in various endurance sports settings. While it has been proposed that cfDNA could be a suitable marker for player load in intermittent sports, the effects on cfDNA of repeated sprinting as an essential feature in intermittent sports are unknown. For the first time, we assessed both alterations of cfDNA due to repeated maximal sprints and due to a professional football game. Nine participants were subjected to a standardised sprint training session with cross-over design of five maximal sprints of 40 meters with either "short" (1 minute) or "long" pauses (5 minutes). Capillary cfDNA and lactate were measured after every sprint and venous cfDNA before and after each series of sprints. Moreover, capillary cfDNA and lactate values were taken in 23 professional football players before and after incremental exercise testing, during the course of a training week at rest (baseline) and in all 17 enrolled players following a season game. Lactate and venous cfDNA increased more pronounced during "short" compared to "long" (1.4-fold, p = 0.032 and 1.7-fold, p = 0.016) and cfDNA correlated significantly with lactate (r = 0.69; psports. In contrast to the potential of more established blood-based markers like IL-6, CK, or CRP, cfDNA shows by far the strongest fold-change and a high correlation with a particular load related aspect in professional football.

  15. Circulating, cell-free DNA as a marker for exercise load in intermittent sports.

    Directory of Open Access Journals (Sweden)

    Nils Haller

    Full Text Available Attempts to establish a biomarker reflecting individual player load in intermittent sports such as football have failed so far. Increases in circulating DNA (cfDNA have been demonstrated in various endurance sports settings. While it has been proposed that cfDNA could be a suitable marker for player load in intermittent sports, the effects on cfDNA of repeated sprinting as an essential feature in intermittent sports are unknown. For the first time, we assessed both alterations of cfDNA due to repeated maximal sprints and due to a professional football game.Nine participants were subjected to a standardised sprint training session with cross-over design of five maximal sprints of 40 meters with either "short" (1 minute or "long" pauses (5 minutes. Capillary cfDNA and lactate were measured after every sprint and venous cfDNA before and after each series of sprints. Moreover, capillary cfDNA and lactate values were taken in 23 professional football players before and after incremental exercise testing, during the course of a training week at rest (baseline and in all 17 enrolled players following a season game.Lactate and venous cfDNA increased more pronounced during "short" compared to "long" (1.4-fold, p = 0.032 and 1.7-fold, p = 0.016 and cfDNA correlated significantly with lactate (r = 0.69; p<0.001. Incremental exercise testing increased cfDNA 7.0-fold (p<0.001. The season game increased cfDNA 22.7-fold (p<0.0001, while lactate showed a 2.0-fold (p = 0.09 increase compared to baseline. Fold-changes in cfDNA correlated with distance covered during game (spearman's r = 0.87, p = 0.0012, while no correlation between lactate and the tracking data could be found.We show for the first time that cfDNA could be an objective marker for distance covered in elite intermittent sports. In contrast to the potential of more established blood-based markers like IL-6, CK, or CRP, cfDNA shows by far the strongest fold-change and a high correlation with a

  16. Intermittent hydronephrosis

    International Nuclear Information System (INIS)

    Knop, J.; Vogel, H.; Hupe, W.

    1981-01-01

    An intermittent hydronephrosis was observed in a 40-year old patient. This disease pattern is due to an incongruity between the formation of urine and the transport capacity in the ureteropelvic junction. The latent impediment of flow becomes manifest with increased urine secretion. Irreversible renal damage can be the result of the repeatedly occurring hydronephrotic crises. (orig.) [de

  17. 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)

  18. 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.

  19. Long term imaging of living brain cancer cells

    Science.gov (United States)

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

    2018-02-01

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

  20. Intermittency '93

    International Nuclear Information System (INIS)

    Bialas, A.

    1993-01-01

    The existing data definitely indicate the existence of intermittency, i.e. of self similar structures in the systems of particles created in high-energy collisions. The effect seems universal: it was found in most of the processes investigated and its measures parameters depend only weakly (if at all) on the process in question. Strong HBT effect was found, suggesting that intermittency is related to space-time structure of the pion source rather than to detailed momentum structure of the production amplitudes. There are indications that this space time structure may be fractal, but more data is needed to establish this. The theoretical explanation remains obscure: it seems that both parton cascade and hadronization play an important role. Their interrelation, however, remains a mystery. 5 figs., 19 refs

  1. Axial tomography in 3D live cell microscopy

    Science.gov (United States)

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

    2017-07-01

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

  2. 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

  3. 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

  4. Longitudinal dynamics of the HIV-specific B cell response during intermittent treatment of primary HIV infection.

    Directory of Open Access Journals (Sweden)

    Godelieve J de Bree

    Full Text Available Neutralizing antibodies develop in natural HIV-1 infection. Their development often takes several years and may rely on chronic virus exposure. At the same time recent studies show that treatment early in infection may provide opportunities for immune preservation. However, it is unknown how intermittent treatment in early infection affects development of the humoral immune response over time. We investigate the effect of cART in early HIV infection on the properties of the memory B cell compartment following 6 months of cART or in the absence of treatment. The patients included participated in the Primo-SHM trial where patients with an early HIV-1 infection were randomized to no treatment or treatment for 24 or 60 weeks.Primo-SHM trial patients selected for the present study were untreated (n = 23 or treated for 24 weeks (n = 24. Here we investigate memory B cell properties at viral set-point and at a late time point (respectively median 54 and 73 weeks before (re-initiation of treatment.At viral set-point, the memory B cell compartment in treated patients demonstrated significantly lower fractions of antigen-primed, activated, memory B cells (p = 0.006. In contrast to untreated patients, in treated patients the humoral HIV-specific response reached a set point over time. At a transcriptional level, sets of genes that showed enhanced expression in memory B cells at viral setpoint in untreated patients, conversely showed rapid increase of expression of the same genes in treated patients at the late time point.These data suggest that, although the memory B cell compartment is phenotypically preserved until viral setpoint after treatment interruption, the development of the HIV-specific antibody response may benefit from exposure to HIV. The effect of viral exposure on B cell properties is also reflected by longitudinal changes in transcriptional profile in memory B cells over time in early treated patients.

  5. Probing the bioelectrochemistry of living cells

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-10-01

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

  6. In-111-oxine red cells for imaging of intermittent G.I. bleeding sites

    International Nuclear Information System (INIS)

    Marcus, C.S.; Angulo, M.C.; Salk, R.D.; Essex, C.E.

    1985-01-01

    Sequential daily abdominal imaging was performed for up to 7 days in 11 patients with intermittent G.I. bleeding after the intravenous administration of lmCi autologous In-111-oxine labeled RBC's. The bleeding sites were identified in 3 patients. The causes were colon carcinoma, diverticulitis, and eroding distal aortic aneutysm. In addition to the imaging information, the authors have obtained preliminary biodistribution and kinetic data on the In-RBC's. Distribution to liver, spleen, and bone marrow was approximately 40%, 40%, and 20%, respectively. (This does not include the quantity of In-111 in the blood pool, which is very high initially and declines with time.) The survival of circulating In-RBC's is described by the equation: Surviving fraction=0.26e/sup -0.0021t/+0.74e/sup -0.00083t/ The halflives of the fast and slow components (x-bar+-x-bar) are 33.4 +- 1.6 hours and 35.0 +- 1.25 days, respectively. The In-oxine label is less stable than Cr-51 but more stable than Tc-99m. At 24 hours, Cr-RBC/In-RBC survival is 1.11 and Cr-RBC/Tc-RBC survival is 1.23. This imaging procedure is quite useful in selected patients

  7. 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.

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

    Science.gov (United States)

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

    2015-05-27

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

  9. 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.

  10. Intermittent PTHrP(1–34) Exposure Augments Chondrogenesis and Reduces Hypertrophy of Mesenchymal Stromal Cells

    Science.gov (United States)

    Fischer, Jennifer; Aulmann, Antje; Dexheimer, Verena; Grossner, Tobias

    2014-01-01

    Phenotype instability and premature hypertrophy prevent the use of human mesenchymal stromal cells (MSCs) for cartilage regeneration. Aim of this study was to investigate whether intermittent supplementation of parathyroid hormone-related protein (PTHrP), as opposed to constant treatment, can beneficially influence MSC chondrogenesis and to explore molecular mechanisms below catabolic and anabolic responses. Human MSCs subjected to chondrogenic induction in high-density culture received PTHrP(1–34), forskolin, dbcAMP, or PTHrP(7–34) either constantly or via 6-h pulses (three times weekly), before proteoglycan, collagen type II, and X deposition; gene expression; and alkaline phosphatase (ALP) activity were assessed. While constant application of PTHrP(1–34) suppressed chondrogenesis of MSCs, pulsed application significantly increased collagen type 2 (COL2A1) gene expression and the collagen type II, proteoglycan, and DNA content of pellets after 6 weeks. Collagen type 10 (COL10A1) gene expression was little affected but Indian hedgehog (IHH) expression and ALP activity were significantly downregulated by pulsed PTHrP. A faster response to PTHrP exposure was recorded for ALP activity over COL2A1 regulation, suggesting that signal duration is critical for catabolic versus anabolic reactions. Stimulation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling by forskolin reproduced major effects of both treatment modes, whereas application of PTHrP(7–34) capable of protein kinase C (PKC) signaling was ineffective. Pulsed PTHrP exposure of MSCs stimulated chondrogenesis and reduced endochondral differentiation apparently uncoupling chondrogenic matrix deposition from hypertrophic marker expression. cAMP/PKA was the major signaling pathway triggering the opposing effects of both treatment modes. Intermittent application of PTHrP represents an important novel means to improve chondrogenesis of MSCs and may be considered as a supporting clinical

  11. 4Pi-confocal microscopy of live cells

    Science.gov (United States)

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

    2002-06-01

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

  12. Frequency and magnitude of intermittent hypoxia modulate endothelial wound healing in a cell culture model of sleep apnea.

    Science.gov (United States)

    Campillo, Noelia; Falcones, Bryan; Montserrat, Josep M; Gozal, David; Obeso, Ana; Gallego-Martin, Teresa; Navajas, Daniel; Almendros, Isaac; Farré, Ramon

    2017-11-01

    Intermittent hypoxia (IH) has been implicated in the cardiovascular consequences of obstructive sleep apnea (OSA). However, the lack of suitable experimental systems has precluded assessment as to whether IH is detrimental, protective, or both for the endothelium. The aim of the work was to determine the effects of frequency and amplitude of IH oxygenation swings on aortic endothelial wound healing. Monolayers of human primary endothelial cells were wounded and subjected to constant oxygenation (1%, 4%, 13%, or 20% O 2 ) or IH at different frequencies (0.6, 6, or 60 cycles/h) and magnitude ranges (13-4% O 2 or 20-1% O 2 ), using a novel well-controlled system, with wound healing being measured after 24 h. Cell monolayer repair was similar at 20% O 2 and 13% O 2 , but was considerably increased (approximately twofold) in constant hypoxia at 4% O 2 The magnitude and frequency of IH considerably modulated wound healing. Cycles ranging 13-4% O 2 at the lowest frequency (0.6 cycles/h) accelerated endothelial wound healing by 102%. However, for IH exposures consisting of 20% to 1% O 2 oscillations, wound closure was reduced compared with oscillation in the 13-4% range (by 74% and 44% at 6 cycles/h and 0.6 cycles/h, respectively). High-frequency IH patterns simulating severe OSA (60 cycles/h) did not significantly modify endothelial wound closure, regardless of the oxygenation cycle amplitude. In conclusion, the frequency and magnitude of hypoxia cycling in IH markedly alter wound healing responses and emerge as key factors determining how cells will respond in OSA. NEW & NOTEWORTHY Intermittent hypoxia (IH) induces cardiovascular consequences in obstructive sleep apnea (OSA) patients. However, the vast array of frequencies and severities of IH previously employed in OSA-related experimental studies has led to controversial results on the effects of IH. By employing an optimized IH experimental system here, we provide evidence that the frequency and magnitude of IH

  13. 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.

  14. Quantification of cytoskeletal deformation in living cells

    NARCIS (Netherlands)

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

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

  15. 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 ...

  16. Fluorescence Dynamics in the Endoplasmic Reticulum of a Live Cell: Time-Resolved Confocal Microscopy.

    Science.gov (United States)

    Ghosh, Shirsendu; Nandi, Somen; Ghosh, Catherine; Bhattacharyya, Kankan

    2016-09-19

    Fluorescence dynamics in the endoplasmic reticulum (ER) of a live non-cancer lung cell (WI38) and a lung cancer cell (A549) are studied by using time-resolved confocal microscopy. To selectively study the organelle, ER, we have used an ER-Tracker dye. From the emission maximum (λmaxem) of the ER-Tracker dye, polarity (i.e. dielectric constant, ϵ) in the ER region of the cells (≈500 nm in WI38 and ≈510 nm in A549) is estimated to be similar to that of chloroform (λmaxem =506 nm, ϵ≈5). The red shift by 10 nm in λmaxem in the cancer cell (A549) suggests a slightly higher polarity compared to the non-cancer cell (WI38). The fluorescence intensity of the ER-Tracker dye exhibits prolonged intermittent oscillations on a timescale of 2-6 seconds for the cancer cell (A549). For the non-cancer cell (WI38), such fluorescence oscillations are much less prominent. The marked fluorescence intensity oscillations in the cancer cell are attributed to enhanced calcium oscillations. The average solvent relaxation time () of the ER region in the lung cancer cell (A549, 250±50 ps) is about four times faster than that in the non-cancer cell (WI38, 1000±50 ps). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Directory of Open Access Journals (Sweden)

    Victoria V. Roshchina

    2012-01-01

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

  18. Intermittent hypoxia hypobaric exposure minimized oxidative stress and antioxidants in brain cells of Sprague Dawleymice

    Directory of Open Access Journals (Sweden)

    Wardaya Wardaya

    2013-05-01

    Full Text Available AbstrakLatar belakang: Hipoksia hypobaric meningkatkan produksi radikal bebas, terutama spesies oksigen reaktif (ROS. Peningkatan ROS akan menyebabkan stres oksidatif bila tidak disertai dengan peningkatan enzim antioksidan. Kondisi ini dapat dikurangi dengan hipoksia hipobarik intermiten (HHI. Tujuan penelitian ini mengidentifikasi frekuensi IHH yang dapat meminimalkan efek hipoksia hipobarik terhadap stres oksidatif dan aktivitas antioksidan spesifik pada tikus Sprague Dawley.Metode: Penelitian eksperimental pada bulan Februari-April 2010, Subjek terdiri dari satu kelompok kontrol dan empat kelompok paparan pada mencit jantan Sprague Dawley. Setiap kelompok terdiri dari 5 tikus. Kelompok kontrol tidak terpapar IHH. Kelompok terpapar (dengan selang waktu satu minggu terpapar sekali, dua kali, tiga kali, atau empat kali IHH. Semua kelompok paparan dipaparkan hipobarik setara dengan ketinggian: 35.000 ft (1 menit, 25.000 ft (5 menit, dan 18.000 ft (25 menit. Jaringan otak diperiksa untuk 8-OHdG dan SOD.Hasil:Setelah tiga paparan IHH tingkat 8-OHdG sudah kembali ke nilai kontrol (P = 0,843. Tingkat SOD meningkat secara progresif pada dua, tiga, dan empat kali paparan IHH. Bahkan setelah paparan kedua, tingkat SOD sudah sama dengan nilai kontrol, 0,231 ± 0,042 (P = 0,191.Kesimpulan: Tiga kali IHH sudah dapat meminimalkan pengaruh hipoksia hipobarik terhadap stres oksidatif dan aktivitas spesifik antioksidan pada tikus Sprague Dawley.Kata kunci: hipoksia hipobarik intermiten, stres oksidatif, antioksidanAbstractBackground: Hypoxia hypobaric increase the production of free radicals, especially reactive oxygen species (ROS. The increase in ROS would cause oxidative stress when not accompanied by an increase in antioxidant enzymes. This condition may minimize by intermittent hypobaric hypoxia (IHH. This study aimed to identify the number of IHH which may minimize the effect of hypoxia hypobaric on oxidative stress and the specific activity of

  19. Intermittent hyperthyreosis

    International Nuclear Information System (INIS)

    Sulman, F.G.; Tal, E.; Pfeifer, Y.; Superstine, E.

    1975-01-01

    Intermittent hyperthyreosis occurs under various forms of stress, especially heat stress. The clinician may diagnose such cases as masked or apathetic hyperthyroidism or 'forme fruste' hyperthyreosis or thyroid autonomy. As most routine and standard tests may here yield inconsistent results, it is the patients' anamnesis which may provide the clue. Our Bioclimatology Unit has now seen over 100 cases in which thyroid hypersensitivity towards heat was the most prominent syndrome: 10-15% of weather-sensitive patients are affected. The patients complain before or during heat spells of such contradictory symptoms as insomnia, irritability, tension, tachycardia, palpitations, precordial pain, dyspnoe, flushes with sweating or chills, tremor, abdominal pain or diarrhea, polyuria or pollakisuria, weight loss in spite of ravenous appetite, fatigue, exhaustion, depression, adynamia, lack of concentration and confusion. Determination of urinary neurohormones allows a differential diagnosis, intermittent hyperthyreosis being characterized by three cardinal symptoms: tachycardia - every case with more than 80 pulse beats being suspect (not specific); urinary histamine - every case excreting more than 90 μg/day being suspect. Again the drawback of this test is its lack of specificity, as histamine may also be increased in cases of allergy and spondylitis; urinary thyroxine - every case excreting more than 20 μg/day T-4 being suspect. This is the only specific test. Therapy should make use of lithium carbonate and betablockers. Propyl thiouracil is rarely required. (orig.) [de

  20. 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.

  1. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-12-15

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

  3. 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

  4. Intermittent Hypoxia and Stem Cell Implants Preserve Breathing Capacity in a Rodent Model of Amyotrophic Lateral Sclerosis

    Science.gov (United States)

    Nichols, Nicole L.; Gowing, Genevieve; Satriotomo, Irawan; Nashold, Lisa J.; Dale, Erica A.; Suzuki, Masatoshi; Avalos, Pablo; Mulcrone, Patrick L.; McHugh, Jacalyn

    2013-01-01

    Rationale: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1G93A rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. Objectives: To preserve or restore phrenic nerve activity in SOD1G93A rats at disease end stage. Methods: SOD1G93A rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. Measurements and Main Results: The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. Conclusions: AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS. PMID:23220913

  5. Intermittent, low dose carbon monoxide exposure enhances survival and dopaminergic differentiation of human neural stem cells

    DEFF Research Database (Denmark)

    Dreyer-Andersen, Nanna; Almeida, Ana Sofia; Jensen, Pia

    2018-01-01

    cells constitute an alternative source of cells for transplantation in Parkinson's disease, but efficient protocols for controlled dopaminergic differentiation need to be developed. Short-term, low-level carbon monoxide (CO) exposure has been shown to affect signaling in several tissues, resulting...... in both protection and generation of reactive oxygen species. The present study investigated the effect of CO produced by a novel CO-releasing molecule on dopaminergic differentiation of human neural stem cells. Short-term exposure to 25 ppm CO at days 0 and 4 significantly increased the relative content...... of β-tubulin III-immunoreactive immature neurons and tyrosine hydroxylase expressing catecholaminergic neurons, as assessed 6 days after differentiation. Also the number of microtubule associated protein 2-positive mature neurons had increased significantly. Moreover, the content of apoptotic cells...

  6. PeakForce Tapping resolves individual microvilli on living cells.

    Science.gov (United States)

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

    2016-02-01

    Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip-sample interactions in time (microseconds) and controlling force in the low pico-Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM-Live Cell probe, having a short cantilever with a 17-µm-long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico-Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions. © 2015 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.

  7. Intermittent IL-7 Signaling Essential for T cell Homeostasis | Center for Cancer Research

    Science.gov (United States)

    In order for the immune system to mount an appropriate response to foreign antigens throughout a person’s life, the body must maintain a sufficient population of circulating mature, naïve T cells, a process known as T cell homeostasis. Previous studies revealed that this process depends upon signaling from the cytokine interleukin-7 (IL-7) as well as from the T cell antigen receptor (TCR). Intriguingly, signals from each pathway affect the other and lead to their alternating activation: IL-7 binding to its receptor leads to increasing expression of the TCR co-receptor CD8; sufficient CD8 expression allows TCRs to signal when bound to self-ligands, blocking IL-7 signaling; suppressed IL-7 signals lead to down-regulation of CD8 and ligand disengagement, which allows T cells to again respond to IL-7. Alfred Singer, M.D., and his colleagues in CCR’s Experimental Immunology Branch set out to understand how this intricate pathway promotes T cell survival.

  8. 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.

  9. Intermittent contact of fluidized anode particles containing exoelectrogenic biofilms for continuous power generation in microbial fuel cells

    KAUST Repository

    Liu, Jia

    2014-09-01

    Current generation in a microbial fuel cell can be limited by the amount of anode surface area available for biofilm formation, and slow substrate degradation kinetics. Increasing the anode surface area can increase the amount of biofilm, but performance will improve only if the anode material is located near the cathode to minimize solution internal resistance. Here we demonstrate that biofilms do not have to be in constant contact with the anode to produce current in an MFC. Granular activated carbon particles enriched with exoelectrogenic biofilm are fluidized (by stirring) in the anode chamber of the MFC, resulting in only intermittent contact between the particles and the anode current collector. The maximum power density generated is 951 ± 10 mW m-2, compared to 813 ± 2 mW m-2 for the control without stirring (packed bed), and 525 ± 1 mW m-2 in the absence of GAC particles and without stirring. GAC-biofilm particles demonstrate capacitor-like behavior, but achieve nearly constant discharge conditions due to the large number of particles that contact the current collector. These results provide proof of concept for the development of flowable electrode reactors, where anode biofilms can be electrically charged in a separate storage tank and then rapidly discharged in compact anode chambers. © 2014 Elsevier B.V. All rights reserved.

  10. Intermittent contact of fluidized anode particles containing exoelectrogenic biofilms for continuous power generation in microbial fuel cells

    KAUST Repository

    Liu, Jia; Zhang, Fang; He, Weihua; Zhang, Xiaoyuan; Feng, Yujie; Logan, Bruce E.

    2014-01-01

    Current generation in a microbial fuel cell can be limited by the amount of anode surface area available for biofilm formation, and slow substrate degradation kinetics. Increasing the anode surface area can increase the amount of biofilm, but performance will improve only if the anode material is located near the cathode to minimize solution internal resistance. Here we demonstrate that biofilms do not have to be in constant contact with the anode to produce current in an MFC. Granular activated carbon particles enriched with exoelectrogenic biofilm are fluidized (by stirring) in the anode chamber of the MFC, resulting in only intermittent contact between the particles and the anode current collector. The maximum power density generated is 951 ± 10 mW m-2, compared to 813 ± 2 mW m-2 for the control without stirring (packed bed), and 525 ± 1 mW m-2 in the absence of GAC particles and without stirring. GAC-biofilm particles demonstrate capacitor-like behavior, but achieve nearly constant discharge conditions due to the large number of particles that contact the current collector. These results provide proof of concept for the development of flowable electrode reactors, where anode biofilms can be electrically charged in a separate storage tank and then rapidly discharged in compact anode chambers. © 2014 Elsevier B.V. All rights reserved.

  11. 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)

  12. Intermittent, low dose carbon monoxide exposure enhances survival and dopaminergic differentiation of human neural stem cells

    DEFF Research Database (Denmark)

    Dreyer-Andersen, Nanna; Almeida, Ana Sofia; Jensen, Pia

    2018-01-01

    Exploratory studies using human fetal tissue have suggested that intrastriatal transplantation of dopaminergic neurons may become a future treatment for patients with Parkinson's disease. However, the use of human fetal tissue is compromised by ethical, regulatory and practical concerns. Human stem...... cells constitute an alternative source of cells for transplantation in Parkinson's disease, but efficient protocols for controlled dopaminergic differentiation need to be developed. Short-term, low-level carbon monoxide (CO) exposure has been shown to affect signaling in several tissues, resulting...... in Parkinson's disease....

  13. [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.

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

    Science.gov (United States)

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

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

  15. Information management for high content live cell imaging

    Directory of Open Access Journals (Sweden)

    White Michael RH

    2009-07-01

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

  16. The live cell irradiation and observation setup at SNAKE

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-06-15

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

  17. 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.

  18. Imaging Proteolysis by Living Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Mansoureh Sameni

    2000-01-01

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

  19. 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...

  20. 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...

  1. 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.

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

    Science.gov (United States)

    Deitch, Arline D.; Moses, Montrose J.

    1957-01-01

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

  3. 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.).

  4. Enhancement of Intermittent Androgen Ablation Therapy by Finasteride Administration in Animal Models

    National Research Council Canada - National Science Library

    Wang, Zhou

    2006-01-01

    .... Intermittent androgen ablation therapy (IAAT) may slow down the development of androgen refractory tumors because intermittent recovery of androgens can induce differentiation of prostatic epithelial cells...

  5. Enhancement of Intermittent Androgen Ablation Therapy by Finasteride Administration in Animal Models

    National Research Council Canada - National Science Library

    Wang, Zhou

    2004-01-01

    .... Intermittent androgen ablation therapy may slow down the development of androgen refractory tumors because intermittent recovery of androgens can induce differentiation of prostatic epithelial cells...

  6. Enhancement of Intermittent Androgen Ablation Therapy by Finasteride Administration in Animal Models

    National Research Council Canada - National Science Library

    Wang, Zhou

    2005-01-01

    .... Intermittent androgen ablation therapy may slow down the development of androgen refractory tumors because intermittent recovery of androgens can induce differentiation of prostatic epithelial cells...

  7. Enhancement of Intermittent Androgen Ablation Therapy by Finasteride Administration in Animal Models

    National Research Council Canada - National Science Library

    Wang, Zhou

    2003-01-01

    .... Intermittent androgen ablation therapy may slow down the development of androgen refractory tumors because intermittent recovery of androgens can induce differentiation of prostatic epithelial cells...

  8. 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\\

  9. 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.

  10. Raman spectroscopy for grading of live osteosarcoma cells.

    Science.gov (United States)

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

    2015-04-18

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

  11. 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...

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

    DEFF Research Database (Denmark)

    Liesche, Johannes; Schulz, Alexander

    2015-01-01

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

  13. 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

  14. Fuel cell/electrochemical capacitor hybrid for intermittent high power applications

    Energy Technology Data Exchange (ETDEWEB)

    Jarvis, L P; Atwater, T B; Cygan, P J [Army Communications-Electronics Command (CECOM), Fort Monmouth, NJ (United States). Research and Development Center

    1999-05-01

    A hybrid power source was demonstrated to successfully power a simulated power load encountered in portable military electronics and communications equipment. The hybrid system consisted of a 25 W proton exchange membrane fuel cell (PEMFC) stack connected in parallel with a 70 F capacitor bank. The cyclic regime of 18.0 W for 2 min followed by 2.5 W for 18 min was chosen as the baseline for the simulation of power load. The operating potential cut-off voltage for pass/failure was set to 3.0 V. At room temperature (23-25 C), the PEMFC alone could not handle the described baseline regime with the PEMFC operating potential dropping below the cut-off voltage within 10 s. The hybrid, however, continuously powered the same regime for 25 h. Its operating potential never reached the voltage cut-off point, not even during the high load of 18.0 W. The tests with hybrid configuration were aborted after 25 h of operation with no signs of output degradation, suggesting that further extended operation was possible. (orig.)

  15. Peripheral blood mononuclear cells HIV DNA levels impact intermittently on neurocognition.

    Directory of Open Access Journals (Sweden)

    Lucette A Cysique

    Full Text Available To determine the contribution of peripheral blood mononuclear cells' (PBMCs HIV DNA levels to HIV-associated dementia (HAD and non-demented HIV-associated neurocognitive disorders (HAND in chronically HIV-infected adults with long-term viral suppression on combined antiretroviral treatment (cART.Eighty adults with chronic HIV infection on cART (>97% with plasma and CSF HIV RNA <50 copies/mL were enrolled into a prospective observational cohort and underwent assessments of neurocognition and pre-morbid cognitive ability at two visits 18 months apart. HIV DNA in PBMCs was measured by real-time PCR at the same time-points.At baseline, 46% had non-demented HAND; 7.5% had HAD. Neurocognitive decline occurred in 14% and was more likely in those with HAD (p<.03. Low pre-morbid cognitive ability was uniquely associated with HAD (p<.05. Log10 HIV DNA copies were stable between study visits (2.26 vs. 2.22 per 106 PBMC. Baseline HIV DNA levels were higher in those with lower pre-morbid cognitive ability (p<.04, and higher in those with no ART treatment during HIV infection 1st year (p = .03. Baseline HIV DNA was not associated with overall neurocognition. However, % ln HIV DNA change was associated with decline in semantic fluency in unadjusted and adjusted analyses (p = .01-.03, and motor-coordination (p = .02-.12 to a lesser extent.PBMC HIV DNA plays a role in HAD pathogenesis, and this is moderated by pre-morbid cognitive ability in the context of long-term viral suppression. While the HIV DNA levels in PBMC are not associated with current non-demented HAND, increasing HIV DNA levels were associated with a decline in neurocognitive functions associated with HAND progression.

  16. Poly (ADP-ribose polymerase plays an important role in intermittent hypoxia-induced cell death in rat cerebellar granule cells

    Directory of Open Access Journals (Sweden)

    Chiu Sheng-Chun

    2012-03-01

    Full Text Available Abstract Background Episodic cessation of airflow during sleep in patients with sleep apnea syndrome results in intermittent hypoxia (IH. Our aim was to investigate the effects of IH on cerebellar granule cells and to identify the mechanism of IH-induced cell death. Methods Cerebellar granule cells were freshly prepared from neonatal Sprague-Dawley rats. IH was created by culturing the cerebellar granule cells in the incubators with oscillating O2 concentration at 20% and 5% every 30 min for 1-4 days. The results of this study are based on image analysis using a confocal microscope and associated software. Cellular oxidative stress increased with increase in IH. In addition, the occurrence of cell death (apoptosis and necrosis increased as the duration of IH increased, but decreased in the presence of an iron chelator (phenanthroline or poly (ADP-ribose polymerase (PARP inhibitors [3-aminobenzamide (3-AB and DPQ]. The fluorescence of caspase-3 remained the same regardless of the duration of IH, and Western blots did not detect activation of caspase-3. However, IH increased the ratio of apoptosis-inducing factor (AIF translocation to the nucleus, while PARP inhibitors (3-AB reduced this ratio. Results According to our findings, IH increased oxidative stress and subsequently leading to cell death. This effect was at least partially mediated by PARP activation, resulting in ATP depletion, calpain activation leading to AIF translocation to the nucleus. Conclusions We suggest that IH induces cell death in rat primary cerebellar granule cells by stimulating oxidative stress PARP-mediated calpain and AIF activation.

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

    Science.gov (United States)

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

    2017-08-01

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

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

    Science.gov (United States)

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

    2017-08-01

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

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

    Science.gov (United States)

    Li, Junjie; Cheng, Ji-Xin

    2014-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Y. L. Gong

    2013-06-01

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

  1. 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.

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

    Science.gov (United States)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2018-04-18

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

  4. 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.

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

    Science.gov (United States)

    Ishida, Takeshi

    2014-04-01

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

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

    Science.gov (United States)

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

    2009-08-13

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

  7. 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.

  8. 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

  9. Digital photocontrol of the network of live excitable cells

    Science.gov (United States)

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

    2011-11-01

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2015-09-08

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

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

    Science.gov (United States)

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

    2016-12-09

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

  13. Detecting protein-protein interactions in living cells

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  14. Raman microscopy of individual living human embryonic stem cells

    Science.gov (United States)

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

    2010-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Saera Hihara

    2012-12-01

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

  16. Automated analysis of invadopodia dynamics in live cells

    Directory of Open Access Journals (Sweden)

    Matthew E. Berginski

    2014-07-01

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

  17. Fluorescent tags of protein function in living cells.

    Science.gov (United States)

    Whitaker, M

    2000-02-01

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

  18. 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.

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

    KAUST Repository

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

    2016-01-01

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

  20. 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.

  1. Effects of Intermittent Administration of Parathyroid Hormone (1-34 on Bone Differentiation in Stromal Precursor Antigen-1 Positive Human Periodontal Ligament Stem Cells

    Directory of Open Access Journals (Sweden)

    Xiaoxiao Wang

    2016-01-01

    Full Text Available Periodontitis is the most common cause of tooth loss and bone destruction in adults worldwide. Human periodontal ligament stem cells (hPDLSCs may represent promising new therapeutic biomaterials for tissue engineering applications. Stromal precursor antigen-1 (STRO-1 has been shown to have roles in adherence, proliferation, and multipotency. Parathyroid hormone (PTH has been shown to enhance proliferation in osteoblasts. Therefore, in this study, we aimed to compare the functions of STRO-1(+ and STRO-1(− hPDLSCs and to investigate the effects of PTH on the osteogenic capacity of STRO-1(+ hPDLSCs in order to evaluate their potential applications in the treatment of periodontitis. Our data showed that STRO-1(+ hPDLSCs expressed higher levels of the PTH-1 receptor (PTH1R than STRO-1(− hPDLSCs. In addition, intermittent PTH treatment enhanced the expression of PTH1R and osteogenesis-related genes in STRO-1(+ hPDLSCs. PTH-treated cells also exhibited increased alkaline phosphatase activity and mineralization ability. Therefore, STRO-1(+ hPDLSCs represented a more promising cell resource for biomaterials and tissue engineering applications. Intermittent PTH treatment improved the capacity for STRO-1(+ hPDLSCs to repair damaged tissue and ameliorate the symptoms of periodontitis.

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

    Directory of Open Access Journals (Sweden)

    Stefanie Galbán

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

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

    Science.gov (United States)

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

    1980-01-01

    The laser dye rhodamine 123 is shown to be a specific probe for the localization of mitochondria in living cells. By virtue of its selectivity for mitochondria and its fluorescent properties, the detectability of mitochondria stained with rhodamine 123 is significantly improved over that provided by conventional light microscopic techniques. With the use of rhodamine 123, it is possible to detect alterations in mitochondrial distribution following transformation by Rous sarcoma virus and changes in the shape and organization of mitochondria induced by colchicine treatment. Images PMID:6965798

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

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

    Science.gov (United States)

    2013-08-14

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

  6. 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

  7. 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.

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

    Science.gov (United States)

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

    2015-05-01

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

  9. 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.

  10. Direct imaging of APP proteolysis in living cells.

    Science.gov (United States)

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

    2017-01-01

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

  11. 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.

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

    Science.gov (United States)

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

    2016-03-01

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

  13. 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

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

    Science.gov (United States)

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

    2016-05-06

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  16. 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.

  17. Lineage tracing in the adult mouse corneal epithelium supports the limbal epithelial stem cell hypothesis with intermittent periods of stem cell quiescence

    Directory of Open Access Journals (Sweden)

    Natalie J. Dorà

    2015-11-01

    Full Text Available The limbal epithelial stem cell (LESC hypothesis proposes that LESCs in the corneal limbus maintain the corneal epithelium both during normal homeostasis and wound repair. The alternative corneal epithelial stem cell (CESC hypothesis proposes that LESCs are only involved in wound repair and CESCs in the corneal epithelium itself maintain the corneal epithelium during normal homeostasis. We used tamoxifen-inducible, CreER-loxP lineage tracing to distinguish between these hypotheses. Clones of labelled cells were induced in adult CAGG-CreER;R26R-LacZ reporter mice and their distributions analysed after different chase periods. Short-lived clones, derived from labelled transient amplifying cells, were shed during the chase period and long-lived clones, derived from stem cells, expanded. At 6 weeks, labelled clones appeared at the periphery, extended centripetally as radial stripes and a few reached the centre by 14 weeks. Stripe numbers depended on the age of tamoxifen treatment. Stripes varied in length, some were discontinuous, few reached the centre and almost half had one end at the limbus. Similar stripes extended across the cornea in CAGG-CreER;R26R-mT/mG reporter mice. The distributions of labelled clones are inconsistent with the CESC hypothesis and support the LESC hypothesis if LESCs cycle between phases of activity and quiescence, each lasting several weeks.

  18. Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kyotani, Yoji, E-mail: cd147@naramed-u.ac.jp [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Department of Pharmacy, Nara Medical University Hospital, Kashihara 634-8522 (Japan); Ota, Hiroyo [Second Department of Internal Medicine, Nara Medical University School of Medicine, Kashihara 634-8522 (Japan); Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Itaya-Hironaka, Asako; Yamauchi, Akiyo; Sakuramoto-Tsuchida, Sumiyo [Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Zhao, Jing; Ozawa, Kentaro; Nagayama, Kosuke; Ito, Satoyasu [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Takasawa, Shin [Department of Biochemistry, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan); Kimura, Hiroshi [Second Department of Internal Medicine, Nara Medical University School of Medicine, Kashihara 634-8522 (Japan); Uno, Masayuki [Department of Pharmacy, Nara Medical University Hospital, Kashihara 634-8522 (Japan); Yoshizumi, Masanori [Department of Pharmacology, Nara Medical University School of Medicine, Kashihara 634-8521 (Japan)

    2013-11-15

    Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation. - Highlights: ●In vitro system for intermittent hypoxia (IH) and sustained hypoxia (SH). ●IH, but not SH, induces the proliferation of rat vascular smooth muscle cell. ●Epiregulin m

  19. Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

    International Nuclear Information System (INIS)

    Kyotani, Yoji; Ota, Hiroyo; Itaya-Hironaka, Asako; Yamauchi, Akiyo; Sakuramoto-Tsuchida, Sumiyo; Zhao, Jing; Ozawa, Kentaro; Nagayama, Kosuke; Ito, Satoyasu; Takasawa, Shin; Kimura, Hiroshi; Uno, Masayuki; Yoshizumi, Masanori

    2013-01-01

    Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation. - Highlights: ●In vitro system for intermittent hypoxia (IH) and sustained hypoxia (SH). ●IH, but not SH, induces the proliferation of rat vascular smooth muscle cell. ●Epiregulin m

  20. Intermittent search strategies

    Science.gov (United States)

    Bénichou, O.; Loverdo, C.; Moreau, M.; Voituriez, R.

    2011-01-01

    This review examines intermittent target search strategies, which combine phases of slow motion, allowing the searcher to detect the target, and phases of fast motion during which targets cannot be detected. It is first shown that intermittent search strategies are actually widely observed at various scales. At the macroscopic scale, this is, for example, the case of animals looking for food; at the microscopic scale, intermittent transport patterns are involved in a reaction pathway of DNA-binding proteins as well as in intracellular transport. Second, generic stochastic models are introduced, which show that intermittent strategies are efficient strategies that enable the minimization of search time. This suggests that the intrinsic efficiency of intermittent search strategies could justify their frequent observation in nature. Last, beyond these modeling aspects, it is proposed that intermittent strategies could also be used in a broader context to design and accelerate search processes.

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

    Science.gov (United States)

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

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

    Science.gov (United States)

    Honigmann, Alf; Nadler, André

    2018-01-09

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

  3. Structural model of radiation effects in living cells

    International Nuclear Information System (INIS)

    Neyman, J.; Puri, P.S.

    1976-01-01

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

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

    Science.gov (United States)

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

    2017-12-01

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

  5. 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)

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-02-01

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

  8. 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.

  9. 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

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

    Science.gov (United States)

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

    2018-03-23

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

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

    Science.gov (United States)

    Nikitin, V A

    2007-01-01

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

  12. Intermittency in branching models

    International Nuclear Information System (INIS)

    Chiu, C.B.; Texas Univ., Austin; Hwa, R.C.; Oregon Univ., Eugene

    1990-01-01

    The intermittency properties of three branching models have been investigated. The factorial moments show power-law behavior as function of small rapidity width. The slopes and energy dependences reveal different characteristics of the models. The gluon model has the weakest intermittency. (orig.)

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

    Czech Academy of Sciences Publication Activity Database

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

    2014-01-01

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

  14. 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)

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    Bu-er Wang

    2015-05-01

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

  17. 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

  18. Mechanics of Cellulose Synthase Complexes in Living Plant Cells

    Science.gov (United States)

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

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

  19. 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.

  20. Intermittent degradation and schizotypy

    Directory of Open Access Journals (Sweden)

    Matthew W. Roché

    2015-06-01

    Full Text Available Intermittent degradation refers to transient detrimental disruptions in task performance. This phenomenon has been repeatedly observed in the performance data of patients with schizophrenia. Whether intermittent degradation is a feature of the liability for schizophrenia (i.e., schizotypy is an open question. Further, the specificity of intermittent degradation to schizotypy has yet to be investigated. To address these questions, 92 undergraduate participants completed a battery of self-report questionnaires assessing schizotypy and psychological state variables (e.g., anxiety, depression, and their reaction times were recorded as they did so. Intermittent degradation was defined as the number of times a subject’s reaction time for questionnaire items met or exceeded three standard deviations from his or her mean reaction time after controlling for each item’s information processing load. Intermittent degradation scores were correlated with questionnaire scores. Our results indicate that intermittent degradation is associated with total scores on measures of positive and disorganized schizotypy, but unrelated to total scores on measures of negative schizotypy and psychological state variables. Intermittent degradation is interpreted as potentially derivative of schizotypy and a candidate endophenotypic marker worthy of continued research.

  1. Prognostic relevance of sunitinib toxicities and comparison of continuous vs. intermittent sunitinib dosing schedule in metastatic renal cell cancer patients

    Directory of Open Access Journals (Sweden)

    Çetin Ordu

    2016-06-01

    Full Text Available Aim of the study : Sunitinib-related side effects may develop as a result of the pharmacokinetic pathway affects the of the drug. Material and methods : Data on mRCC patients were obtained from the hospital archives. Outcomes of patients were evaluated in terms of related prognostic factors, sunitinib adverse events during the treatment, and two different sunitinib dosing schedules. Results : Seventy patients diagnosed with mRCC and treated with sunitinib were analyzed for prognostic factors and survival rates. During the mean follow-up of 33.5 months, 38 (54% patients were alive and 32 (46% patients died. The median time of overall survival (OS and progression-free survival (PFS was 27 months (12–61 and 19 months (5–45, respectively. In univariate analysis, good prognostic risk group according to the Memorial Sloan-Kettering Cancer Center (MSKCC, hypothyroidism as sunitinib toxicity and patients on sunitinib treatment more than 1 year were favorable prognostic factors for OS. Leukopenia and fatigue as sunitinib toxicity were poor prognostic factors for OS. PFS and OS of the patients were not significantly different when we compared intermittent (4/2 vs. continuous treatment dosing schedules. Conclusions : As a result of this trial, having hypothyroidism as an adverse effect of sunitinib was a favorable prognostic factor for OS and PFS in mRCC patients. It was also found that 4/2 and continuous dosing schedules of sunitinib did not give rise to different outcomes in mRCC patients.

  2. Acute effects of resistance exercise and intermittent intense aerobic exercise on blood cell count and oxidative stress in trained middle-aged women

    Energy Technology Data Exchange (ETDEWEB)

    Cardoso, A.M. [Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Bagatini, M.D. [Curso de Enfermagem, Campus Chapecó, Universidade Federal da Fronteira Sul, Chapecó, SC (Brazil); Roth, M.A. [Departamento de Desportos Individuais, Centro de Educação Física e Desportos, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Martins, C.C.; Rezer, J.F.P. [Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Mello, F.F. [Departamento de Desportos Individuais, Centro de Educação Física e Desportos, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Lopes, L.F.D. [Departamento de Administração, Centro de Ciências Sociais e Humanas, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Morsch, V.M.; Schetinger, M.R.C. [Departamento de Química, Centro de Ciências Naturais e Exatas, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil)

    2012-10-26

    The aim of this study was to compare the effect of an intermittent intense aerobic exercise session and a resistance exercise session on blood cell counts and oxidative stress parameters in middle-aged women. Thirty-four women were selected and divided into three groups: RE group (performing 60 min of resistance exercises, N = 12), spinning group (performing 60 min of spinning, N = 12), and control group (not exercising regularly, N = 10). In both exercise groups, lymphocytes and monocytes decreased after 1-h recuperation (post-exercise) compared to immediately after exercise (P < 0.05). Immediately after exercise, in both exercised groups, a significant increase in TBARS (from 16.5 ± 2 to 25 ± 2 for the spinning group and from 18.6 ± 1 to 28.2 ± 3 nmol MDA/mL serum for the RE group) and protein carbonyl (from 1.0 ± 0.3 to 1.6 ± 0.2 for the spinning group and from 0.9 ± 0.2 to 1.5 ± 0.2 nmol/mg protein for the RE group) was observed (P < 0.05). A decrease in antioxidant activities (non-protein sulfhydryl, superoxide dismutase, catalase) was also demonstrated with a negative correlation between damage markers and antioxidant body defenses (P < 0.05). These results indicate that an acute bout of intermittent or anaerobic exercise induces immune suppression and increases the production of reactive oxygen species, causing oxidative stress in middle-aged and trained women. Furthermore, we demonstrated that trained women show improved antioxidant capacity and lower oxidative damage than sedentary ones, demonstrating the benefits of chronic regular physical activity.

  3. Acute effects of resistance exercise and intermittent intense aerobic exercise on blood cell count and oxidative stress in trained middle-aged women

    Directory of Open Access Journals (Sweden)

    A.M. Cardoso

    2012-12-01

    Full Text Available The aim of this study was to compare the effect of an intermittent intense aerobic exercise session and a resistance exercise session on blood cell counts and oxidative stress parameters in middle-aged women. Thirty-four women were selected and divided into three groups: RE group (performing 60 min of resistance exercises, N = 12, spinning group (performing 60 min of spinning, N = 12, and control group (not exercising regularly, N = 10. In both exercise groups, lymphocytes and monocytes decreased after 1-h recuperation (post-exercise compared to immediately after exercise (P < 0.05. Immediately after exercise, in both exercised groups, a significant increase in TBARS (from 16.5 ± 2 to 25 ± 2 for the spinning group and from 18.6 ± 1 to 28.2 ± 3 nmol MDA/mL serum for the RE group and protein carbonyl (from 1.0 ± 0.3 to 1.6 ± 0.2 for the spinning group and from 0.9 ± 0.2 to 1.5 ± 0.2 nmol/mg protein for the RE group was observed (P < 0.05. A decrease in antioxidant activities (non-protein sulfhydryl, superoxide dismutase, catalase was also demonstrated with a negative correlation between damage markers and antioxidant body defenses (P < 0.05. These results indicate that an acute bout of intermittent or anaerobic exercise induces immune suppression and increases the production of reactive oxygen species, causing oxidative stress in middle-aged and trained women. Furthermore, we demonstrated that trained women show improved antioxidant capacity and lower oxidative damage than sedentary ones, demonstrating the benefits of chronic regular physical activity.

  4. Acute effects of resistance exercise and intermittent intense aerobic exercise on blood cell count and oxidative stress in trained middle-aged women

    International Nuclear Information System (INIS)

    Cardoso, A.M.; Bagatini, M.D.; Roth, M.A.; Martins, C.C.; Rezer, J.F.P.; Mello, F.F.; Lopes, L.F.D.; Morsch, V.M.; Schetinger, M.R.C.

    2012-01-01

    The aim of this study was to compare the effect of an intermittent intense aerobic exercise session and a resistance exercise session on blood cell counts and oxidative stress parameters in middle-aged women. Thirty-four women were selected and divided into three groups: RE group (performing 60 min of resistance exercises, N = 12), spinning group (performing 60 min of spinning, N = 12), and control group (not exercising regularly, N = 10). In both exercise groups, lymphocytes and monocytes decreased after 1-h recuperation (post-exercise) compared to immediately after exercise (P < 0.05). Immediately after exercise, in both exercised groups, a significant increase in TBARS (from 16.5 ± 2 to 25 ± 2 for the spinning group and from 18.6 ± 1 to 28.2 ± 3 nmol MDA/mL serum for the RE group) and protein carbonyl (from 1.0 ± 0.3 to 1.6 ± 0.2 for the spinning group and from 0.9 ± 0.2 to 1.5 ± 0.2 nmol/mg protein for the RE group) was observed (P < 0.05). A decrease in antioxidant activities (non-protein sulfhydryl, superoxide dismutase, catalase) was also demonstrated with a negative correlation between damage markers and antioxidant body defenses (P < 0.05). These results indicate that an acute bout of intermittent or anaerobic exercise induces immune suppression and increases the production of reactive oxygen species, causing oxidative stress in middle-aged and trained women. Furthermore, we demonstrated that trained women show improved antioxidant capacity and lower oxidative damage than sedentary ones, demonstrating the benefits of chronic regular physical activity

  5. 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.

  6. Intermittent Explosive Disorder

    Directory of Open Access Journals (Sweden)

    Lut Tamam

    2011-09-01

    Full Text Available Intermittent explosive disorder is an impulse control disorder characterized by the occurrence of discrete episodes of failure to resist aggressive impulses that result in violent assault or destruction of property. Though the prevalence intermittent explosive disorder has been reported to be relatively rare in frontier studies on the field, it is now common opinion that intermittent explosive disorder is far more common than previously thought especially in clinical psychiatry settings. Etiological studies displayed the role of both psychosocial factors like childhood traumas and biological factors like dysfunctional neurotransmitter systems and genetics. In differential diagnosis of the disorder, disorders involving agression as a symptom such as alcohol and drug intoxication, antisocial and borderline personality disorders, personality changes due to general medical conditions and behavioral disorder should be considered. A combination of pharmacological and psychotherapeutic approaches are suggested in the treatment of the disorder. This article briefly reviews the historical background, diagnostic criteria, epidemiology, etiology and treatment of intermittent explosive disorder.

  7. Intermittent heating of buildings

    Energy Technology Data Exchange (ETDEWEB)

    Kohonen, K

    1983-02-01

    Conditions for intermittent heating of buildings are considered both theoretically and experimentally. Thermal behaviour of buildings adn rooms in intermittent heating is simulated by a program based on the convective heat balance equation and by simplified RC-models. The preheat times and the heating energy savings compared with continuous heating are presented for typical lightweight, mediumweight and heavyweight classroom and office modules. Formulaes for estimating the oversizing of the radiator network, the maximum heat output of heat exchangers in district heating and the efficiency of heating boilers in intermittent heating are presented. The preheat times and heating energy savings with different heating control systems are determined also experimentally in eight existing buildings. In addition some principles for the planning and application of intermittent heating systems are suggested.

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

    Science.gov (United States)

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

    2017-09-01

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

  9. Optimal intermittent search strategies

    International Nuclear Information System (INIS)

    Rojo, F; Budde, C E; Wio, H S

    2009-01-01

    We study the search kinetics of a single fixed target by a set of searchers performing an intermittent random walk, jumping between different internal states. Exploiting concepts of multi-state and continuous-time random walks we have calculated the survival probability of a target up to time t, and have 'optimized' (minimized) it with regard to the transition probability among internal states. Our model shows that intermittent strategies always improve target detection, even for simple diffusion states of motion

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

    Science.gov (United States)

    De, Abhijit; Gambhir, Sanjiv Sam

    2005-12-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-08-04

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

  13. 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

  14. Damage of Inner Ear Sensory Hair Cells via Mitochondrial Loss in a Murine Model of Sleep Apnea With Chronic Intermittent Hypoxia.

    Science.gov (United States)

    Seo, Young Joon; Ju, Hyun Mi; Lee, Sun Hee; Kwak, Sang Hyun; Kang, Min Jung; Yoon, Joo-Heon; Kim, Chang-Hoon; Cho, Hyung-Ju

    2017-09-01

    Investigating the exact pathophysiology of obstructive sleep apnea syndrome (OSAS)-induced hearing loss is critical. We sought to verify the hypothesis that a correlation exists between mitochondrial dysfunction in inner ear hair cells and the auditory dysfunction induced by chronic intermittent hypoxia (CIH) in a murine model of sleep apnea. C57BL/6J adult male mice were randomized to 4 weeks of CIH (n = 12) or normoxia (Sham) (n = 12). Hearing threshold was determined by auditory brainstem response. The activity of mitochondria was compared between CIH and Sham mice. Histological assessment and transmission electron microscopy were performed for assessing morphologic changes in mitochondria. The number of mtDNA copies as well as the levels of PGC1-α, Tfam, and VDAC (voltage-dependent anion channel) were determined in the hair cells of CIH mice. We observed that hearing ability in CIH mice was impaired and hair-cell mitochondria in CIH mice were fewer compared to that in Sham and also displayed an aberrant morphology. The mRNA levels of PGC-1α and Tfam were higher in the CIH group than in the Sham group. Moreover, the expression of VDAC was increased in the tectorial membrane, the basilar membrane, and especially in the inner hair cells of CIH mice. This study using CIH mice as a model for OSAS provides evidence of an association between OSAS and auditory function alteration, as well as of mitochondria being part of the pathophysiology of hearing impairment. Further investigation is required to determine whether mitochondria could serve as a valid target for preventive or therapeutic purposes. © Sleep Research Society 2017. Published by Oxford University Press on behalf of the Sleep Research Society. All rights reserved. For permissions, please e-mail journals.permissions@oup.com.

  15. 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.

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

    National Research Council Canada - National Science Library

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

    2006-01-01

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

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

    Science.gov (United States)

    Carlson, Alicia L; Fujisaki, Joji; Wu, Juwell; Runnels, Judith M; Turcotte, Raphaël; Spencer, Joel A; Celso, Cristina Lo; Scadden, David T; Strom, Terry B; Lin, Charles P

    2013-01-01

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

  18. Intermittent Hypoxia Is Associated With High Hypoxia Inducible Factor-1α but Not High Vascular Endothelial Growth Factor Cell Expression in Tumors of Cutaneous Melanoma Patients

    Directory of Open Access Journals (Sweden)

    Isaac Almendros

    2018-04-01

    Full Text Available Epidemiological associations linking between obstructive sleep apnea and poorer solid malignant tumor outcomes have recently emerged. Putative pathways proposed to explain that these associations have included enhanced hypoxia inducible factor (HIF-1α and vascular endothelial growth factor (VEGF cell expression in the tumor and altered immune functions via intermittent hypoxia (IH. Here, we examined relationships between HIF-1α and VEGF expression and nocturnal IH in cutaneous melanoma (CM tumor samples. Prospectively recruited patients with CM tumor samples were included and underwent overnight polygraphy. General clinical features, apnea–hypopnea index (AHI, desaturation index (DI4%, and CM characteristics were recorded. Histochemical assessments of VEGF and HIF-1α were performed, and the percentage of positive cells (0, <25, 25–50, 51–75, >75% was blindly tabulated for VEGF expression, and as 0, 0–5.9, 6.0–10.0, >10.0% for HIF-1α expression, respectively. Cases with HIF-1α expression >6% (high expression were compared with those <6%, and VEGF expression >75% of cells was compared with those with <75%. 376 patients were included. High expression of VEGF and HIF-1α were seen in 88.8 and 4.2% of samples, respectively. High expression of VEGF was only associated with increasing age. However, high expression of HIF-1α was significantly associated with age, Breslow index, AHI, and DI4%. Logistic regression showed that DI4% [OR 1.03 (95% CI: 1.01–1.06] and Breslow index [OR 1.28 (95% CI: 1.18–1.46], but not AHI, remained independently associated with the presence of high HIF-1α expression. Thus, IH emerges as an independent risk factor for higher HIF-1α expression in CM tumors and is inferentially linked to worse clinical CM prognostic indicators.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  20. 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.

  1. Honokiol protects pancreatic β cell against high glucose and intermittent hypoxia-induced injury by activating Nrf2/ARE pathway in vitro and in vivo.

    Science.gov (United States)

    Li, Chen-Guang; Ni, Chang-Lin; Yang, Min; Tang, Yun-Zhao; Li, Zhu; Zhu, Yan-Juan; Jiang, Zhen-Huan; Sun, Bei; Li, Chun-Jun

    2018-01-01

    Obstructive sleep apnea hypopnea syndrome (OSAHS) is associated with glucose intolerance, insulin resistance and type 2 diabetes mellitus (T2DM). Although several studies have revealed that intermittent hypoxia (IH) in OSAHS may further aggravate pancreatic β cell damage and promote the evolution of type 2 diabetes (T2DM) by increasing oxidative stress, the underlying mechanisms are unclear. Honokiol, a potent radical scavenger, has been demonstrated to ameliorate oxidative stress in many cases. The present study aimed to explore the potential mechanism of IH and diabetes synergistically damage and destruct the pancreatic β cell, examine the effects of honokiol on ameliorating pancreatic β cell injury in this context and explore the mechanism of such effects. High glucose (HG) cultured INS-1 cells were exposed to 50 μM of honokiol for 24, 48 and 72 h with or without IH intervention. T2DM rats were treated with honokiol and exposed to 80 s of IH followed by 160 s of normoxia for 8 weeks. The cell proliferation, apoptosis and oxidative stress were measured. Blood glucose, insulin, glucagon and HOMA-IR (Homeostasis model assessment -insulin resistence) were also detected, and the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were detected by immunofluorescence staining and western blotting. Honokiol can reduce oxidative stress, cytotoxicity and apoptosis in the INS-1 cells of rats receiving HG treatment or both HG and IH treatment. IH can further aggravate pancreas dysfunction, cause a marked elevation in fasting blood glucose, glucagon, HOMA-IR and oxidative stress levels in DM rats. In addition, honokiol can effectively activate the Nrf2/ARE pathway and reverse this pancreatic dysfunction in vivo and in vitro. These findings indicate that honokiol acts as a potent ROS scavenger via Nrf2/ARE pathway and effectively attenuates oxidative stress and improves pancreatic β cell function of DM rats under IH

  2. Acute effects of resistance exercise and intermittent intense aerobic exercise on blood cell count and oxidative stress in trained middle-aged women.

    Science.gov (United States)

    Cardoso, A M; Bagatini, M D; Roth, M A; Martins, C C; Rezer, J F P; Mello, F F; Lopes, L F D; Morsch, V M; Schetinger, M R C

    2012-12-01

    The aim of this study was to compare the effect of an intermittent intense aerobic exercise session and a resistance exercise session on blood cell counts and oxidative stress parameters in middle-aged women. Thirty-four women were selected and divided into three groups: RE group (performing 60 min of resistance exercises, N = 12), spinning group (performing 60 min of spinning, N = 12), and control group (not exercising regularly, N = 10). In both exercise groups, lymphocytes and monocytes decreased after 1-h recuperation (post-exercise) compared to immediately after exercise (P exercise, in both exercised groups, a significant increase in TBARS (from 16.5 ± 2 to 25 ± 2 for the spinning group and from 18.6 ± 1 to 28.2 ± 3 nmol MDA/mL serum for the RE group) and protein carbonyl (from 1.0 ± 0.3 to 1.6 ± 0.2 for the spinning group and from 0.9 ± 0.2 to 1.5 ± 0.2 nmol/mg protein for the RE group) was observed (P exercise induces immune suppression and increases the production of reactive oxygen species, causing oxidative stress in middle-aged and trained women. Furthermore, we demonstrated that trained women show improved antioxidant capacity and lower oxidative damage than sedentary ones, demonstrating the benefits of chronic regular physical activity.

  3. Safety, adherence and acceptability of intermittent tenofovir/emtricitabine as HIV pre-exposure prophylaxis (PrEP among HIV-uninfected Ugandan volunteers living in HIV-serodiscordant relationships: a randomized, clinical trial.

    Directory of Open Access Journals (Sweden)

    Freddie M Kibengo

    Full Text Available BACKGROUND: Efficacy of oral pre-exposure prophylaxis (PrEP in prevention of HIV acquisition has been evaluated using a daily regimen. However, adherence to long term daily medication is rarely perfect. Intermittent regimen may be a feasible alternative. Preclinical studies have demonstrated effectiveness of intermittent PrEP in SHIV prevention among animals. However, little is known about intermittent PrEP regimens. DESIGN: Seventy two HIV-uninfected volunteers in HIV serodiscordant couple relationships in Uganda were randomly assigned to receive daily oral Tenofovir/Emtricitabine (TDF/FTC-Truvada or placebo, or intermittent (Monday, Friday and within 2 hours after sex, not to exceed one dose per day oral TDF/FTC or placebo in a 2:1:2:1 ratio. Volunteers and study staff were blinded to drug assignment, but not to regimen assignment. METHODS: Volunteers were followed for 4 months after randomization, with monthly clinical and laboratory safety assessments and comprehensive HIV risk reduction services. Adherence was monitored using medication event monitoring system (MEMS and self-report. Sexual activity data were collected via daily short text message (SMS and self-report. HIV-specific immune responses were assessed by IFN-γ ELISPOT. RESULTS: Both daily and intermittent oral TDF/FTC regimens were well tolerated. Median MEMS adherence rates were 98% (IQR: 93-100 for daily PrEP regimen, 91% (IQR: 73-97 for fixed intermittent dosing and 45% (IQR: 20-63 for post-coital dosing. SMS response rate was 74%, but increased to 80% after excluding server outages; results may have been affected by the novelty of this measure. The majority of volunteers expressed willingness with no particular preference for either regimen. CONCLUSIONS: Both daily and intermittent oral PrEP dosing regimens were safe. Adherence was high for daily and fixed intermittent dosing; post-coital dosing was associated with poor adherence. Fixed intermittent PrEP regimens may be

  4. 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.

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

    International Nuclear Information System (INIS)

    So, Min-kyung; Yao Hequan; Rao Jianghong

    2008-01-01

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

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

    OpenAIRE

    sprotocols

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-02-11

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

  8. Optimal intermittent search strategies

    Energy Technology Data Exchange (ETDEWEB)

    Rojo, F; Budde, C E [FaMAF, Universidad Nacional de Cordoba, Ciudad Universitaria, X5000HUA Cordoba (Argentina); Wio, H S [Instituto de Fisica de Cantabria, Universidad de Cantabria and CSIC E-39005 Santander (Spain)

    2009-03-27

    We study the search kinetics of a single fixed target by a set of searchers performing an intermittent random walk, jumping between different internal states. Exploiting concepts of multi-state and continuous-time random walks we have calculated the survival probability of a target up to time t, and have 'optimized' (minimized) it with regard to the transition probability among internal states. Our model shows that intermittent strategies always improve target detection, even for simple diffusion states of motion.

  9. Intermittency and random matrices

    Science.gov (United States)

    Sokoloff, Dmitry; Illarionov, E. A.

    2015-08-01

    A spectacular phenomenon of intermittency, i.e. a progressive growth of higher statistical moments of a physical field excited by an instability in a random medium, attracted the attention of Zeldovich in the last years of his life. At that time, the mathematical aspects underlying the physical description of this phenomenon were still under development and relations between various findings in the field remained obscure. Contemporary results from the theory of the product of independent random matrices (the Furstenberg theory) allowed the elaboration of the phenomenon of intermittency in a systematic way. We consider applications of the Furstenberg theory to some problems in cosmology and dynamo theory.

  10. 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.

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

    Science.gov (United States)

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

    2013-07-21

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

  12. 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.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  14. 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

  15. 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

  16. 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.

  17. A self-powered biosensing device with an integrated hybrid biofuel cell for intermittent monitoring of analytes.

    Science.gov (United States)

    Majdecka, Dominika; Draminska, Sylwia; Janusek, Dariusz; Krysinski, Paweł; Bilewicz, Renata

    2018-04-15

    In this work, we propose an integrated self-powered sensing system, driven by a hybrid biofuel cell (HBFC) with carbon paper discs coated with multiwalled carbon nanotubes. The sensing system has a biocathode made from laccase or bilirubin oxidase, and the anode is made from a zinc plate. The system includes a dedicated custom-built electronic control unit for the detection of oxygen and catechol analytes, which are central to medical and environmental applications. Both the HBFC and sensors, operate in a mediatorless direct electron transfer mode. The measured characteristics of the HBFC with externally applied resistance included the power-time dependencies under flow cell conditions, the sensors performance (evaluated by cyclic voltammetry), and chronoamperometry. The HBFC is integrated with analytical devices and operating in a pulse mode form long-run monitoring experiments. The HBFC generated sufficient power for wireless data transmission to a local computer. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Detection and analysis of apoptosis- and autophagy-related miRNAs of mouse vascular endothelial cells in chronic intermittent hypoxia model.

    Science.gov (United States)

    Liu, Kai-Xiong; Chen, Gong-Ping; Lin, Ping-Li; Huang, Jian-Chai; Lin, Xin; Qi, Jia-Chao; Lin, Qi-Chang

    2018-01-15

    Endothelial dysfunction is the main pathogenic mechanism of cardiovascular complications induced by obstructive sleep apnea/hyponea syndrome (OSAHS). Chronic intermittent hypoxia (CIH) is the primary factor of OSAHS-associated endothelial dysfunction. The hypoxia inducible factor (HIF) pathway regulates the expression of downstream target genes and mediates cell apoptosis caused by CIH-induced endothelial injury. miRNAs play extensive and important negative regulatory roles in this process at the post-transcriptional level. However, the regulatory mechanism of miRNAs in CIH tissue models remains unclear. The present study established a mouse aortic endothelial cell model of CIH in an attempt to screen out specific miRNAs by using miRNA chip analysis. It was found that 14 miRNAs were differentially expressed. Of them, 6 were significantly different and verified by quantitative real-time PCR (Q-PCR), of which four were up-regulated and two were down-regulated markedly. To gain an unbiased global perspective on subsequent regulation by altered miRNAs, we established signaling networks by GO to predict the target genes of the 6 miRNAs. It was found that the 6 identified miRNAs were apoptosis- or autophagy-related target genes. Down-regulation of miR-193 inhibits CIH induced endothelial injury and apoptosis- or autophagy-related protein expression. In conclusion, our results showed that CIH could induce differential expression of miRNAs, and alteration in the miRNA expression pattern was associated with the expression of apoptosis- or autophagy-related genes. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2013-01-01

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

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    Paolo Cinelli

    2010-03-01

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

  2. 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

  3. Discarded cell phone lithium ion batteries state of health quick method analysis by galvanostatic intermittent titration technique (GITT concept

    Directory of Open Access Journals (Sweden)

    Paulo Rogério Catarini

    2009-03-01

    Full Text Available The state of health (SOH is a important evaluation parameter to rechargeable batteries, because determine its cycle life and help on electric devices supplied by batteries maintenance. In this work the lithium ion discards cell phones batteries state of health and apparent diffusion coefficient (Dap were measured and correlated which purpose is diminish the batteries analyze time. The apparent diffusion coefficient is a ionic diffusion coefficient modification from GITT technique. The SOH and Dap correlation is well behaved, disclosing a cubic dependency. The time analyze was reduced by more than 1 h.

  4. Intermittent Testicular Torsion

    African Journals Online (AJOL)

    2017-06-02

    Jun 2, 2017 ... had prior episodes of testicular pain, suggesting that they may have had intermittent torsion before .... None of the patients had antecedent history of sexual exposure, fever, or urinary tract infection .... torsion of the spermatic cord portends an increased risk of acute testicular infarction. J Urol 2008;180 4 ...

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

    Science.gov (United States)

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

    2010-03-01

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

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

    Science.gov (United States)

    Peng, Tao; Hang, Howard C

    2016-11-02

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

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

    Science.gov (United States)

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

    2015-01-15

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

  8. 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.

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

  12. 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....

  13. 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.

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

    Science.gov (United States)

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

    2003-12-03

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

  15. 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.

  16. Hollow fiber: a biophotonic implant for live cells

    Science.gov (United States)

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

    2009-02-01

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

  17. Combination chemotherapy with intermittent erlotinib and pemetrexed for pretreated patients with advanced non-small cell lung cancer: a phase I dose-finding study

    International Nuclear Information System (INIS)

    Minami, Seigo; Tachibana, Isao; Komuta, Kiyoshi; Kawase, Ichiro; Kijima, Takashi; Takahashi, Ryo; Kida, Hiroshi; Nakatani, Takeshi; Hamaguchi, Masanari; Takeuchi, Yoshiko; Nagatomo, Izumi; Yamamoto, Suguru

    2012-01-01

    Erlotinib and pemetrexed have been approved for the second-line treatment of non-small cell lung cancer (NSCLC). These two agents have different mechanisms of action. Combined treatment with erlotinib and pemetrexed could potentially augment the antitumor activity of either agent alone. In the present study, we investigated the safety profile of combined administration of the two agents in pretreated NSCLC patients. A phase I dose-finding study (Trial registration: UMIN000002900) was performed in patients with stage III/IV nonsquamous NSCLC whose disease had progressed on or after receiving first-line chemotherapy. Patients received 500 mg/m 2 of pemetrexed intravenously every 21 days and erlotinib (100 mg at Level 1 and 150 mg at Level 2) orally on days 2–16. Twelve patients, nine males and three females, were recruited. Patient characteristics included a median age of 66 years (range, 48–78 years), stage IV disease (nine cases), adenocarcinoma (seven cases) and activating mutation-positives in the epidermal growth factor receptor gene (two cases). Treatment was well-tolerated, and the recommended dose of erlotinib was fixed at 150 mg. Dose-limiting toxicities were experienced in three patients and included: grade 3 elevation of serum alanine aminotransferase, repetitive grade 4 neutropenia that required reduction of the second dose of pemetrexed and grade 3 diarrhea. No patient experienced drug-induced interstitial lung disease. Three patients achieved a partial response and stable disease was maintained in five patients. Combination chemotherapy of intermittent erlotinib with pemetrexed was well-tolerated, with promising efficacy against pretreated advanced nonsquamous NSCLC

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

    Directory of Open Access Journals (Sweden)

    Huiqiang Wang

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  20. 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.

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

    KAUST Repository

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

    2016-01-01

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

  2. 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.

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  4. 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

  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. Visualizing how cancer chromosome abnormalities form in living cells

    Science.gov (United States)

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

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

    Directory of Open Access Journals (Sweden)

    Thomas Näsström

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

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

    Directory of Open Access Journals (Sweden)

    Neil O Carragher

    2011-04-01

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

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

    Science.gov (United States)

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

    2009-06-01

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

  11. 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

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

    Science.gov (United States)

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

    2014-03-28

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

  13. 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.

  14. 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

  15. 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.

  16. Quasistatic Dynamics with Intermittency

    International Nuclear Information System (INIS)

    Leppänen, Juho; Stenlund, Mikko

    2016-01-01

    We study an intermittent quasistatic dynamical system composed of nonuniformly hyperbolic Pomeau–Manneville maps with time-dependent parameters. We prove an ergodic theorem which shows almost sure convergence of time averages in a certain parameter range, and identify the unique physical family of measures. The theorem also shows convergence in probability in a larger parameter range. In the process, we establish other results that will be useful for further analysis of the statistical properties of the model.

  17. Quasistatic Dynamics with Intermittency

    Energy Technology Data Exchange (ETDEWEB)

    Leppänen, Juho; Stenlund, Mikko, E-mail: mikko.stenlund@helsinki.fi [University of Helsinki, Department of Mathematics and Statistics (Finland)

    2016-06-15

    We study an intermittent quasistatic dynamical system composed of nonuniformly hyperbolic Pomeau–Manneville maps with time-dependent parameters. We prove an ergodic theorem which shows almost sure convergence of time averages in a certain parameter range, and identify the unique physical family of measures. The theorem also shows convergence in probability in a larger parameter range. In the process, we establish other results that will be useful for further analysis of the statistical properties of the model.

  18. 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.

  19. LONG-LIVED BONE MARROW PLASMA CELLS DURING IMMUNE RESPONSE TO ALPHA (1→3 DEXTRAN

    Directory of Open Access Journals (Sweden)

    I. N. Chernyshova

    2015-01-01

    Full Text Available Production kinetics and some functional properties of long-lived marrow plasma cells were studied in mice immunized with T-independent type 2 antigens. Alpha (1→3 dextran was used as an antigen for immunization. The mice were immunized by dextran, and the numbers of IgM antibody producing cells were determined by ELISPOT method. The cell phenotype was determined by cytofluorimetric technique. In the area of normal bone marrow lymphocytes ~4% of T and ~85% of B cells were detected. About 35% of the cells expressed a plasmocyte marker (CD138; 3% were CD138+IgM+, and about 6% of the lymphocytes were double-positive for CD138+IgA+. Among spleen lymphocytes, 50% of T and 47% of B cells were detected. About 1.5% lymphocytes were CD138+, and 0.5% were positive for CD138 and IgM. Time kinetics of antibody-producing cells in bone marrow and spleen was different. In spleen populations, the peak amounts of antibody-secreting cells have been shown on the day 4; the process abated by the day 28. Vice versa, the numbers of the antibody-producing cells in bone marrow started to increase on the day 4. The process reached its maximum on day 14, and after 28th day became stationary. The in vitro experiments have shown that supplementation of bone marrow cells from immune mice with dextran did not influence their functional activity. It was previously shown for cells responding to T-dependent antigens only. A specific marker for the long-lived plasma cells is still unknown. However, these cells possess a common CD138 marker specific for all plasma cells. A method for isolation of bone marrow CD138+ cells was developed. The CD138+ cells were of 87-97% purity, being enriched in long-lived bone marrow cells, and produced monospecific antibodies.

  20. 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

  1. Quantitative live imaging of endogenous DNA replication in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Andrew Burgess

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

  2. A minimal rupture cascade model for living cell plasticity

    Science.gov (United States)

    Polizzi, Stefano; Laperrousaz, Bastien; Perez-Reche, Francisco J.; Nicolini, Franck E.; Maguer Satta, Véronique; Arneodo, Alain; Argoul, Françoise

    2018-05-01

    Under physiological and pathological conditions, cells experience large forces and deformations that often exceed the linear viscoelastic regime. Here we drive CD34+ cells isolated from healthy and leukemic bone marrows in the highly nonlinear elasto-plastic regime, by poking their perinuclear region with a sharp AFM cantilever tip. We use the wavelet transform mathematical microscope to identify singular events in the force-indentation curves induced by local rupture events in the cytoskeleton (CSK). We distinguish two types of rupture events, brittle failures likely corresponding to irreversible ruptures in a stiff and highly cross-linked CSK and ductile failures resulting from dynamic cross-linker unbindings during plastic deformation without loss of CSK integrity. We propose a stochastic multiplicative cascade model of mechanical ruptures that reproduces quantitatively the experimental distributions of the energy released during these events, and provides some mathematical and mechanistic understanding of the robustness of the log-normal statistics observed in both brittle and ductile situations. We also show that brittle failures are relatively more prominent in leukemia than in healthy cells suggesting their greater fragility.

  3. 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.

  4. 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.

  5. Live Cell Refractometry Using Hilbert Phase Microscopy and Confocal Reflectance Microscopy†

    Science.gov (United States)

    Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

    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

  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. 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)

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

    Directory of Open Access Journals (Sweden)

    Andrea Brown Forrester

    2015-09-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

  10. 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.

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

    DEFF Research Database (Denmark)

    Ilieva, Mirolyuba

    2013-01-01

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

  12. 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.

  13. 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.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

  15. Optofluidics for handling and analysis of single living cells

    KAUST Repository

    Perozziello, Gerardo; Candeloro, Patrizio; Coluccio, Maria Laura; Di Fabrizio, Enzo M.

    2017-01-01

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

  16. Relationships between Cargo, Cell Penetrating Peptides and Cell Type for Uptake of Non-Covalent Complexes into Live Cells

    Directory of Open Access Journals (Sweden)

    Andrea-Anneliese Keller

    2013-02-01

    Full Text Available Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.

  17. 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.

  18. 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%).

  19. 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.

  20. 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

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

    OpenAIRE

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

    1993-01-01

    simple and relatively inexpensive system is described for obtaining quantitative fluorescence measurements on single living cells loaded with a fluorescent probe to study cell physiological processes. The light emitted from the fluorescent cells is captured by and transported through an optical fiber. After passage through appropriate filters the light is measured using a photomultiplier tube. The optical fiber is mounted in one of the microscope outlets. Signals derived from the photomultipl...

  2. 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

  3. 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.

  4. Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

    Science.gov (United States)

    Baltar, Federico

    2018-01-01

    Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles. PMID:29354095

  5. Live attenuated measles virus vaccine therapy for locally established malignant glioblastoma tumor cells

    Directory of Open Access Journals (Sweden)

    Al-Shammari AM

    2014-05-01

    Full Text Available Ahmed M Al-Shammari,1 Farah E Ismaeel,2 Shahlaa M Salih,2 Nahi Y Yaseen11Experimental Therapy Department, Iraqi Center for Cancer and Medical Genetic Researches, Mustansiriya University, 2Departments of Biotechnology, College of Science, Al-Nahrain University, Baghdad, IraqAbstract: Glioblastoma multiforme is the most aggressive malignant primary brain tumor in humans, with poor prognosis. A new glioblastoma cell line (ANGM5 was established from a cerebral glioblastoma multiforme in a 72-year-old Iraqi man who underwent surgery for an intracranial tumor. This study was carried out to evaluate the antitumor effect of live attenuated measles virus (MV Schwarz vaccine strain on glioblastoma multiforme tumor cell lines in vitro. Live attenuated MV Schwarz strain was propagated on Vero, human rhabdomyosarcoma, and human glioblastoma-multiform (ANGM5 cell lines. The infected confluent monolayer appeared to be covered with syncytia with granulation and vacuolation, as well as cell rounding, shrinkage, and large empty space with cell debris as a result of cell lysis and death. Cell lines infected with virus have the ability for hemadsorption to human red blood cells after 72 hours of infection, whereas no hemadsorption of uninfected cells is seen. Detection of MV hemagglutinin protein by monoclonal antibodies in infected cells of all cell lines by immunocytochemistry assay gave positive results (brown color in the cytoplasm of infected cells. Cell viability was measured after 72 hours of infection by 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay. Results showed a significant cytotoxic effect for MV (P≤0.05 on growth of ANGM5 and rhabdomyosarcoma cell lines after 72 hours of infection. Induction of apoptosis by MV was assessed by measuring mitochondrial membrane potentials in tumor cells after 48, 72, and 120 hours of infection. Apoptotic cells were counted, and the mean percentage of dead cells was significantly higher after 48, 72

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

    Science.gov (United States)

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

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

  7. 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.

  8. 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...

  9. 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

  10. Detecting infrared luminescence and non-chemical signaling of living cells: single cell mid-IR spectroscopy in cryogenic environments

    Science.gov (United States)

    Pereverzev, Sergey

    2017-02-01

    Many life-relevant interaction energies are in IR range, and it is reasonable to believe that some biochemical reactions inside cells can results in emission of IR photons. Cells can use this emission for non-chemical and non-electrical signaling. Detecting weak infrared radiation from live cells is complicated because of strong thermal radiation background and absorption of radiation by tissues. A microfluidic device with live cells inside a vacuum cryogenic environment should suppress this background, and thereby permit observation of live cell auto-luminescence or signaling in the IR regime. One can make IR-transparent windows not emitting in this range, so only the cell and a small amount of liquid around it will emit infrared radiation. Currently mid-IR spectroscopy of single cells requires the use of a synchrotron source to measure absorption or reflection spectra. Decreasing of thermal radiation background will allow absorption and reflection spectroscopy of cells without using synchrotron light. Moreover, cell auto-luminescence can be directly measured. The complete absence of thermal background radiation for cryogenically cooled samples allows the use IR photon-sensitive detectors and obtaining single molecule sensitivity in IR photo-luminescence measurements. Due to low photon energies, photo-luminescence measurements will be non-distractive for pressures samples. The technique described here is based upon US patent 9366574.

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

    Science.gov (United States)

    Gadalla, Atef; Dehoux, Thomas; Audoin, Bertrand

    2014-05-01

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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. 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.

  17. Label-free and live cell imaging by interferometric scattering microscopy.

    Science.gov (United States)

    Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

    2018-03-14

    Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

  18. 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

  19. 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.

  20. Accurate live and dead bacterial cell enumeration using flow cytometry (Conference Presentation)

    Science.gov (United States)

    Ou, Fang; McGoverin, Cushla; Swift, Simon; Vanholsbeeck, Frédérique

    2017-03-01

    Flow cytometry (FCM) is based on the detection of scattered light and fluorescence to identify cells with particular characteristics of interest. However most FCM cannot precisely control the flow through its interrogation point and hence the volume and concentration of the sample cannot be immediately obtained. The easiest, most reliable and inexpensive way of obtaining absolute counts with FCM is by using reference beads. We investigated a method of using FCM with reference beads to measure live and dead bacterial concentration over the range of 106 to 108 cells/mL and ratio varying from 0 to 100%. We believe we are the first to use this method for such a large cell concentration range while also establishing the effect of varying the live/dead bacteria ratios. Escherichia coli solutions with differing ratios of live:dead cells were stained with fluorescent dyes SYTO 9 and propidium iodide (PI), which label live and dead cells, respectively. Samples were measured using a LSR II Flow Cytometer (BD Biosciences); using 488 nm excitation with 20 mW power. Both SYTO 9 and PI fluorescence were collected and threshold was set to side scatter. Traditional culture-based plate count was done in parallel to the FCM analysis. The concentration of live bacteria from FCM was compared to that obtained by plate counts. Preliminary results show that the concentration of live bacteria obtained by FCM and plate counts correlate well with each other and indicates this may be extended to a wider concentration range or for studying other cell characteristics.

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  2. 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.

  3. 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

  4. 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.

  5. Atomic force microscopy as a tool for the investigation of living cells.

    Science.gov (United States)

    Morkvėnaitė-Vilkončienė, Inga; Ramanavičienė, Almira; Ramanavičius, Arūnas

    2013-01-01

    Atomic force microscopy is a valuable and useful tool for the imaging and investigation of living cells in their natural environment at high resolution. Procedures applied to living cell preparation before measurements should be adapted individually for different kinds of cells and for the desired measurement technique. Different ways of cell immobilization, such as chemical fixation on the surface, entrapment in the pores of a membrane, or growing them directly on glass cover slips or on plastic substrates, result in the distortion or appearance of artifacts in atomic force microscopy images. Cell fixation allows the multiple use of samples and storage for a prolonged period; it also increases the resolution of imaging. Different atomic force microscopy modes are used for the imaging and analysis of living cells. The contact mode is the best for cell imaging because of high resolution, but it is usually based on the following: (i) image formation at low interaction force, (ii) low scanning speed, and (iii) usage of "soft," low resolution cantilevers. The tapping mode allows a cell to behave like a very solid material, and destructive shear forces are minimized, but imaging in liquid is difficult. The force spectroscopy mode is used for measuring the mechanical properties of cells; however, obtained results strongly depend on the cell fixation method. In this paper, the application of 3 atomic force microscopy modes including (i) contact, (ii) tapping, and (iii) force spectroscopy for the investigation of cells is described. The possibilities of cell preparation for the measurements, imaging, and determination of mechanical properties of cells are provided. The applicability of atomic force microscopy to diagnostics and other biomedical purposes is discussed.

  6. 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.

  7. Analysis of surface properties of fixed and live cells using derivatized agarose beads.

    Science.gov (United States)

    Navarro, Vanessa M; Walker, Sherri L; Badali, Oliver; Abundis, Maria I; Ngo, Lylla L; Weerasinghe, Gayani; Barajas, Marcela; Zem, Gregory; Oppenheimer, Steven B

    2002-01-01

    A novel assay has been developed for the histochemical characterization of surface properties of cells based on their adhesion to agarose beads derivatized with more than 100 types of molecules, including sugars, lectins and other proteins, and amino acids. The assay simply involves mixing small quantities of washed cells and beads in droplets on glass microscope slides and determining to which beads various cell types adhere. Distilled water was found to be the best medium for this assay because added ions or molecules in other media inhibit adhesion in some cases. Many cells, however, cannot tolerate distilled water. Here we show that cells fixed with either of two fixatives (1% formaldehyde or Prefer fixative) displayed similar bead-binding properties as did live cells. Specificity of cell-bead binding was tested by including specific free molecules in the test suspensions in hapten-type inhibition experiments. If a hapten compound inhibited live-cell adhesion to a specific bead, it also inhibited fixed-cell adhesion to a specific bead. The results of these experiments suggest that fixed cells display authentic surface properties, opening the door for the use of this assay with many cell types that cannot tolerate distilled water.

  8. [Non-invasive analysis of proteins in living cells using NMR spectroscopy].

    Science.gov (United States)

    Tochio, Hidehito; Murayama, Shuhei; Inomata, Kohsuke; Morimoto, Daichi; Ohno, Ayako; Shirakawa, Masahiro

    2015-01-01

    NMR spectroscopy enables structural analyses of proteins and has been widely used in the structural biology field in recent decades. NMR spectroscopy can be applied to proteins inside living cells, allowing characterization of their structures and dynamics in intracellular environments. The simplest "in-cell NMR" approach employs bacterial cells; in this approach, live Escherichia coli cells overexpressing a specific protein are subjected to NMR. The cells are grown in an NMR active isotope-enriched medium to ensure that the overexpressed proteins are labeled with the stable isotopes. Thus the obtained NMR spectra, which are derived from labeled proteins, contain atomic-level information about the structure and dynamics of the proteins. Recent progress enables us to work with higher eukaryotic cells such as HeLa and HEK293 cells, for which a number of techniques have been developed to achieve isotope labeling of the specific target protein. In this review, we describe successful use of electroporation for in-cell NMR. In addition, (19)F-NMR to characterize protein-ligand interactions in cells is presented. Because (19)F nuclei rarely exist in natural cells, when (19)F-labeled proteins are delivered into cells and (19)F-NMR signals are observed, one can safely ascertain that these signals originate from the delivered proteins and not other molecules.

  9. Time series modeling of live-cell shape dynamics for image-based phenotypic profiling.

    Science.gov (United States)

    Gordonov, Simon; Hwang, Mun Kyung; Wells, Alan; Gertler, Frank B; Lauffenburger, Douglas A; Bathe, Mark

    2016-01-01

    Live-cell imaging can be used to capture spatio-temporal aspects of cellular responses that are not accessible to fixed-cell imaging. As the use of live-cell imaging continues to increase, new computational procedures are needed to characterize and classify the temporal dynamics of individual cells. For this purpose, here we present the general experimental-computational framework SAPHIRE (Stochastic Annotation of Phenotypic Individual-cell Responses) to characterize phenotypic cellular responses from time series imaging datasets. Hidden Markov modeling is used to infer and annotate morphological state and state-switching properties from image-derived cell shape measurements. Time series modeling is performed on each cell individually, making the approach broadly useful for analyzing asynchronous cell populations. Two-color fluorescent cells simultaneously expressing actin and nuclear reporters enabled us to profile temporal changes in cell shape following pharmacological inhibition of cytoskeleton-regulatory signaling pathways. Results are compared with existing approaches conventionally applied to fixed-cell imaging datasets, and indicate that time series modeling captures heterogeneous dynamic cellular responses that can improve drug classification and offer additional important insight into mechanisms of drug action. The software is available at http://saphire-hcs.org.

  10. Intermittency in Complex Flows

    Science.gov (United States)

    Ben Mahjoub, Otman; Redondo, Jose M.

    2017-04-01

    Experimental results of the complex turbulent wake of a cilinder in 2D [1] and 3D flows [2] were used to investigate the scaling of structure functions, similar research was also performed on wave propagation and breaking in the Ocean [3], in the the stratified Atmosphere (ABL) [4] and in a 100large flume (UPC) for both regular and irregular waves, where long time series of waves propagating and generating breaking turbulence velocity rms and higher order measurements were taken in depth. [3,5] by means of a velocimeter SONTEK3-D. The probability distribution functions of the velocity differences and their non Gaussian distribution related to the energy spectrum indicate that irregularity is an important source of turbulence. From Kolmogorov's K41 and K61 intermittency correction: the p th-order longitudinal velocity structure function δul at scale l in the inertial range of three-dimensional fully developed turbulence is related by ⟨δup⟩ = ⟨(u(x+ l)- u(x))p⟩ ˜ ɛp0/3lp/3 l where ⟨...⟩ represents the spatial average over flow domain, with ɛ0 the mean energy dissipation per unit mass and l is the separation distance. The importance of the random nature of the energy dissipation led to the K62 theory of intermittency, but locality and non-homogeneity are key issues. p p/3 p/3 ξd ⟨δul⟩ ˜ ⟨ɛl ⟩l ˜ l and ξp = p 3 + τp/3 , where now ɛl is a fractal energy dissipation at scale l, τp/3 is the scaling of and ξp is the scaling exponent of the velocity structure function of order p. Both in K41 and K62, the structure functions of third order related to skewness is ξ3 = 1. But this is not true either. We show that scaling exponents ξp do deviate from early studies that only investigated homogeneous turbulence, where a large inertial range dominates. The use of multi-fractal analysis and improvements on Structure function calculations on standard Enhanced mixing is an essential property of turbulence and efforts to alter and to control

  11. Intermittency in nuclear multifragmentation

    International Nuclear Information System (INIS)

    Ploszajczak, M.; Tucholski, A.

    1990-07-01

    Fluctuations of the fragment size distribution in a percolation model and in nuclear multifragmentation following the breakup of high energy nuclei in the nuclear emulsion are studied using the method of scaled factorial moments. An intermittent patern of fluctuations is found in the data as well as in the percolation lattice calculation. This is a consequence of both a self-similarity in the fragment size distribution and a random character for the scaling law. These fluctuations are in general well-described by percolation model. The multifractal dimensions are calculated and their relevance to the study of possible critical behaviour is pointed out. (orig.)

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

    Science.gov (United States)

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

    2014-11-01

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

  13. Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

    Science.gov (United States)

    Hayashi, Takahiro; Hamachi, Itaru

    2012-09-18

    Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional

  14. Nanograting-based plasmon enhancement for total internal reflection fluorescence microscopy of live cells

    International Nuclear Information System (INIS)

    Kim, Kyujung; Cho, Eun-Jin; Suh, Jin-Suck; Huh, Yong-Min; Kim, Donghyun; Kim, Dong Jun

    2009-01-01

    We investigated evanescent field enhancement based on subwavelength nanogratings for improved sensitivity in total internal reflection microscopy of live cells. The field enhancement is associated with subwavelength-grating-coupled plasmon excitation. An optimum sample employed a silver grating on a silver film and an SF10 glass substrate. Field intensity was enhanced by approximately 90% when measured by fluorescent excitation of microbeads relative to that on a bare prism as a control, which is in good agreement with numerical results. The subwavelength-grating-mediated field enhancement was also applied to live cell imaging of quantum dots, which confirmed the sensitivity enhancement qualitatively.

  15. 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. Multidimensional intermittency in hadronic collisions

    International Nuclear Information System (INIS)

    Pan, J.; Hwa, R.C.

    1992-06-01

    The study of intermittency in high-energy hadronic collisions by the Monte Carlo code ECCO is extended to 3-dimensional phase space. Strong intermittency is found in agreement with the data. Fluctuation in the impact parameter is responsible for the intermittency in lnp T , and the transverse-momentum conservation leads to negative intermittency slopes in the azimuthal angle φ. The Ochs-Wosiek plots are linear in all dimensions having universal slopes. An exponent ν = 1.448 emerges to characterize multiparticle production in pp collisions. The properties of G moments are also examined, and the fractal dimensions determined

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

    Science.gov (United States)

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

    2014-01-01

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

  18. 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.

  19. 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.

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

    Science.gov (United States)

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

    2010-09-01

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

  1. 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.

  2. Cell cycle accumulation of the proliferating cell nuclear antigen PCN-1 transitions from continuous in the adult germline to intermittent in the early embryo of C. elegans.

    Science.gov (United States)

    Kocsisova, Zuzana; Kornfeld, Kerry; Schedl, Tim

    2018-05-30

    The proliferating cell nuclear antigen (PCNA or PCN-1 in C. elegans), an essential processivity factor for DNA polymerase δ, has been widely used as a marker of S-phase. In C. elegans early embryos, PCN-1 accumulation is cyclic, localizing to the nucleus during S-phase and the cytoplasm during the rest of the cell cycle. The C. elegans larval and adult germline is an important model systems for studying cell cycle regulation, and it was observed that the cell cycle regulator cyclin E (CYE-1 in C. elegans) displays a non-cyclic, continuous accumulation pattern in this tissue. The accumulation pattern of PCN-1 has not been well defined in the larval and adult germline, and the objective of this study was to determine if the accumulation pattern is cyclic, as in other cells and organisms, or continuous, similar to cyclin E. To study the larval and adult germline accumulation of PCN-1 expressed from its native locus, we used CRISPR/Cas9 technology to engineer a novel allele of pcn-1 that encodes an epitope-tagged protein. S-phase nuclei were labeled using EdU nucleotide incorporation, and FLAG::PCN-1 was detected by antibody staining. All progenitor zone nuclei, including those that were not in S-phase (as they were negative for EdU staining) showed PCN-1 accumulation, indicating that PCN-1 accumulated during all cell cycle phases in the germline progenitor zone. The same result was observed with a GFP::PCN-1 fusion protein expressed from a transgene. pcn-1 loss-of-function mutations were analyzed, and pcn-1 was necessary for robust fertility and embryonic development. In the C. elegans early embryo as well as other organisms, PCN-1 accumulates in nuclei only during S-phase. By contrast, in the progenitor zone of the germline of C. elegans, PCN-1 accumulated in nuclei during all cell cycle stages. This pattern is similar to accumulation pattern of cyclin E. These observations support the model that mitotic cell cycle regulation in the germline stem and progenitor

  3. A Novel Technique to Follow Consequences of Exogenous Factors, Including Therapeutic Drugs, on Living Human Breast Epithelial Cells

    Science.gov (United States)

    1999-07-01

    and lipid vectors, are being tested. Concurrent with the development of procedures for live - cell imaging , we are examining the distribution of proteins...dimensional matrix. These studies have not yet begun. There are a number of procedures that must be developed and perfected in the live - cell imaging , as...components of the Wnt signaling pathway are too preliminary and require additional research prior to publication. (9) CONCLUSIONS Live cell imaging of

  4. Quantitative live-cell imaging of human immunodeficiency virus (HIV-1) assembly.

    Science.gov (United States)

    Baumgärtel, Viola; Müller, Barbara; Lamb, Don C

    2012-05-01

    Advances in fluorescence methodologies make it possible to investigate biological systems in unprecedented detail. Over the last few years, quantitative live-cell imaging has increasingly been used to study the dynamic interactions of viruses with cells and is expected to become even more indispensable in the future. Here, we describe different fluorescence labeling strategies that have been used to label HIV-1 for live cell imaging and the fluorescence based methods used to visualize individual aspects of virus-cell interactions. This review presents an overview of experimental methods and recent experiments that have employed quantitative microscopy in order to elucidate the dynamics of late stages in the HIV-1 replication cycle. This includes cytosolic interactions of the main structural protein, Gag, with itself and the viral RNA genome, the recruitment of Gag and RNA to the plasma membrane, virion assembly at the membrane and the recruitment of cellular proteins involved in HIV-1 release to the nascent budding site.

  5. Raman tweezers spectroscopy of live, single red and white blood cells.

    Directory of Open Access Journals (Sweden)

    Aseefhali Bankapur

    Full Text Available An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC and white blood cells (WBC under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW. Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip.

  6. Live-cell imaging of post-golgi transport vesicles in cultured hippocampal neurons

    DEFF Research Database (Denmark)

    Jensen, Camilla Stampe; Misonou, Hiroaki

    2015-01-01

    compartments of neurons. In the past two decades, the establishment and advancement of fluorescent protein technology have provided us with opportunities to study how proteins are trafficked in living cells. However, live imaging of trafficking processes in neurons necessitate imaging tools to distinguish...... the several different routes that neurons use for protein trafficking. Here we provide a novel protocol to selectively visualize post-Golgi transport vesicles carrying fluorescent-labeled ion channel proteins in living neurons. Further, we provide a number of analytical tools we developed to quantify...... mechanisms by which post-Golgi vesicles are trafficked in neurons. Our protocol uniquely combines the classic temperature-block with close monitoring of the transient expression of transfected protein tagged with fluorescent proteins, and provides a quick and easy way to study protein trafficking in living...

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

    Directory of Open Access Journals (Sweden)

    Hans M Larsson

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

  8. Realignment process of actin stress fibers in single living cells studied by focused femtosecond laser irradiation

    OpenAIRE

    Yasukuni, Ryohei; Spitz, Jean-Alexis; Meallet-Renault, Rachel; Negishi, Takayuki; Tada, Takuji; Hosokawa, Yoichiroh; Asahi, Tsuyoshi; Shukunami, Chisa; Hiraki, Yuji; Masuhara, Hiroshi

    2007-01-01

    Three-dimensional dissection of a single actin stress fiber in a living cell was performed based on multi-photon absorption of a focused femtosecond laser pulse. The realignment process of an actin stress fiber was investigated after its direct cutting by a single-shot femtosecond laser pulse irradiation by high-speed transmission and fluorescence imaging methods. It was confirmed that mechanical force led by the femtosecond laser cutting propagates to entire cell through the cytockelton in a...

  9. 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

  10. A new image correction method for live cell atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Shen, Y; Sun, J L; Zhang, A; Hu, J; Xu, L X [College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200030 (China)

    2007-04-21

    During live cell imaging via atomic force microscopy (AFM), the interactions between the AFM probe and the membrane yield distorted cell images. In this work, an image correction method was developed based on the force-distance curve and the modified Hertzian model. The normal loading and lateral forces exerted on the cell membrane by the AFM tip were both accounted for during the scanning. Two assumptions were made in modelling based on the experimental measurements: (1) the lateral force on the endothelial cells was linear to the height; (2) the cell membrane Young's modulus could be derived from the displacement measurement of a normal force curve. Results have shown that the model could be used to recover up to 30% of the actual cell height depending on the loading force. The accuracy of the model was also investigated with respect to the loading force and mechanical property of the cell membrane.

  11. 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.

  12. Effect of infrared light on live blood cells: Role of β-carotene.

    Science.gov (United States)

    Barkur, Surekha; Bankapur, Aseefhali; Chidangil, Santhosh; Mathur, Deepak

    2017-06-01

    We have utilized Raman tweezers to measure and assign micro-Raman spectra of optically trapped, live red blood cells (RBCs), white blood cells (WBCs) and platelets. Various types of WBCs- both granulocytes, lymphocytes, and their different types have been studied. The Raman bands are assigned to different biomolecules of blood cells. The Raman spectra thus obtained has been enabled detection of β-carotene in these blood cells, the spectral features of which act as a signature that facilitates experimental probing of the effect of 785nm laser light on different blood cells as a function of incident laser power in the mW range. The spectral changes that we obtain upon laser irradiation indicate that, both haemoglobin as well as the cell membrane sustains damage. In case of lymphocytes and platelets the peaks corresponding to β-carotene showed drastic changes. Thorough analysis of the spectral changes indicates possibility of free radical induced damage of β-carotene in lymphocytes and platelets. Among different blood cells, RBCs have a power threshold of only 10mW. The power threshold for other types of blood cells is somewhat higher, but always below about 30mW. These values are likely to serve as useful guides for Raman tweezers based experiments on live cells. Copyright © 2017. Published by Elsevier B.V.

  13. 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.

  14. 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

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

    International Nuclear Information System (INIS)

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

    2017-01-01

    Recent developments in super-resolution microscopy make it possible to resolve structures in biological cells at a spatial resolution of a few nm and observe dynamical processes with a temporal resolution of ms to μ s. However, the optimal structural resolution requires repeated illumination cycles and is thus limited to chemically fixed cells. For live cell applications substantial improvement over classical Abbe-limited imaging can already be obtained in adherent or slow moving cells. Nonetheless, a large group of cells are fast moving and thus could not yet be addressed with live cell super-resolution microscopy. These include flagellate pathogens like African trypanosomes, the causative agents of sleeping sickness in humans and nagana in livestock. Here, we present an embedding method based on a in situ forming cytocompatible UV-crosslinked hydrogel. The fast cross-linking hydrogel immobilizes trypanosomes efficiently to allow microscopy on the nanoscale. We characterized both the trypanosomes and the hydrogel with respect to their autofluorescence properties and found them suitable for single-molecule fluorescence microscopy (SMFM). As a proof of principle, SMFM was applied to super-resolve a structure inside the living trypanosome. We present an image of a flagellar axoneme component recorded by using the intrinsic blinking behavior of eYFP. (paper)

  16. Hoechst tagging: a modular strategy to design synthetic fluorescent probes for live-cell nucleus imaging.

    Science.gov (United States)

    Nakamura, Akinobu; Takigawa, Kazumasa; Kurishita, Yasutaka; Kuwata, Keiko; Ishida, Manabu; Shimoda, Yasushi; Hamachi, Itaru; Tsukiji, Shinya

    2014-06-11

    We report a general strategy to create small-molecule fluorescent probes for the nucleus in living cells. Our strategy is based on the attachment of the DNA-binding Hoechst compound to a fluorophore of interest. Using this approach, simple fluorescein, BODIPY, and rhodamine dyes were readily converted to novel turn-on fluorescent nucleus-imaging probes.

  17. The spatio-temporal organization of DNA-repair: a live cell study

    NARCIS (Netherlands)

    D. Hoogstraten (Deborah)

    2003-01-01

    textabstractThe aim of the work outlined in this thesis is to gain more insight into the organization, dynamic properties and differential reaction kinetics of NER factors within living mammalian cell nuclei. To accomplish this, we made use of the green fluorescent protein technology to study TFIIH

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

    OpenAIRE

    WU, Aibin; DUAN, Liping

    2014-01-01

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

  19. Using in Vitro live-cell imaging to explore chemotherapeutics delivered by lipid-based nanoparticles

    NARCIS (Netherlands)

    A.L.B. Seynhaeve (Ann); T.L.M. ten Hagen (Timo)

    2017-01-01

    textabstractConventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in

  20. Fungicidal mechanisms of cathelicidins LL-37 and CATH-2 revealed by live-cell imaging

    NARCIS (Netherlands)

    Ordonez Alvarez, Soledad; Amarullah, Ilham H; Wubbolts, Richard W; Veldhuizen, Edwin J A; Haagsman, Henk P

    2014-01-01

    Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the

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

    KAUST Repository

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

    2017-01-01

    of the bacterial CRISPR-Cas9 system. By fusing eGFP/mRuby2 to the catalytically inactive version of Streptococcus pyogenes and Staphylococcus aureus Cas9, we show robust visualization of telomere repeats in live leaf cells of Nicotiana benthamiana. By tracking

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

    Science.gov (United States)

    Yerramilli, V Siddartha; Kim, Kyung Hyuk

    2018-03-16

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

  3. Synthesis, biological evaluation, and live cell imaging of novel fluorescent duocarmycin analogs.

    Science.gov (United States)

    Tietze, Lutz F; Behrendt, Frank; Pestel, Galina F; Schuberth, Ingrid; Mitkovski, Mišo

    2012-11-01

    For a better understanding of the mode of action of duocarmycin and its analogs, the novel fluorescent duocarmycin derivatives 13-15 and 17b-19b were synthesized, and their bioactivity as well as their cellular uptake investigated using confocal laser scanning microscopy (CLSM) in live-cell imaging experiments. Copyright © 2012 Verlag Helvetica Chimica Acta AG, Zürich.

  4. Caveolae-mediated endocytosis of biocompatible gold nanoparticles in living Hela cells

    DEFF Research Database (Denmark)

    Hao, Xian; Wu, Jiazhen; Shan, Yuping

    2012-01-01

    the internalization mechanism of small-size AuNPs by living Hela cells. Herein, we found that the caveolae-mediated endocytosis was the dominant pathway for the intracellular delivery of small-size AuNPs. The intracellular delivery was suppressed when we depleted the cholesterol with methyl-β-cyclodextrin (M beta CD...

  5. 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.

  6. 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

  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. Affecting Factors and Outcome on Intermittent Internet Pulling Behavior in Taiwan's Undergraduate Students

    Science.gov (United States)

    Yang, Hui-Jen; Lay, Yun-Long

    2011-01-01

    Nowadays people's lives heavily rely on Internet facilities. Internet users generally have constant Internet connectivity and intermittently click on sites they want to access even amidst studying or working. In this study, we sought to examine the factors affecting intermittent Internet pulling behavior on undergraduate students. Furthermore, the…

  9. Automatic analysis of dividing cells in live cell movies to detect mitotic delays and correlate phenotypes in time.

    Science.gov (United States)

    Harder, Nathalie; Mora-Bermúdez, Felipe; Godinez, William J; Wünsche, Annelie; Eils, Roland; Ellenberg, Jan; Rohr, Karl

    2009-11-01

    Live-cell imaging allows detailed dynamic cellular phenotyping for cell biology and, in combination with small molecule or drug libraries, for high-content screening. Fully automated analysis of live cell movies has been hampered by the lack of computational approaches that allow tracking and recognition of individual cell fates over time in a precise manner. Here, we present a fully automated approach to analyze time-lapse movies of dividing cells. Our method dynamically categorizes cells into seven phases of the cell cycle and five aberrant morphological phenotypes over time. It reliably tracks cells and their progeny and can thus measure the length of mitotic phases and detect cause and effect if mitosis goes awry. We applied our computational scheme to annotate mitotic phenotypes induced by RNAi gene knockdown of CKAP5 (also known as ch-TOG) or by treatment with the drug nocodazole. Our approach can be readily applied to comparable assays aiming at uncovering the dynamic cause of cell division phenotypes.

  10. Correlation between live attenuated measles viral load and growth inhibition percentage in non-small cell lung cancer cell line

    Directory of Open Access Journals (Sweden)

    Rasha Fadhel Obaid

    2018-03-01

    Conclusion Live attenuated measles virus strain induced cytotoxic effect against human lung cancer cell line (A549 by induction of apoptosis as an important mechanism of anti-tumor activity, in addition, it indicates a correlation between the quantity of MV genomesand percentage of growth inhibition. This relation  has proved that measles virus had anticancer effect.

  11. Metabolic Effects of Intermittent Fasting.

    Science.gov (United States)

    Patterson, Ruth E; Sears, Dorothy D

    2017-08-21

    The objective of this review is to provide an overview of intermittent fasting regimens, summarize the evidence on the health benefits of intermittent fasting, and discuss physiological mechanisms by which intermittent fasting might lead to improved health outcomes. A MEDLINE search was performed using PubMed and the terms "intermittent fasting," "fasting," "time-restricted feeding," and "food timing." Modified fasting regimens appear to promote weight loss and may improve metabolic health. Several lines of evidence also support the hypothesis that eating patterns that reduce or eliminate nighttime eating and prolong nightly fasting intervals may result in sustained improvements in human health. Intermittent fasting regimens are hypothesized to influence metabolic regulation via effects on (a) circadian biology, (b) the gut microbiome, and (c) modifiable lifestyle behaviors, such as sleep. If proven to be efficacious, these eating regimens offer promising nonpharmacological approaches to improving health at the population level, with multiple public health benefits.

  12. 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.)

  13. In vivo MRI discrimination between live and lysed iron-labelled cells using balanced steady state free precession

    Energy Technology Data Exchange (ETDEWEB)

    Ribot, E.J. [University of Western Ontario, Imaging Research Laboratories, Robarts Research Institute, London, ON (Canada); Foster, P.J. [University of Western Ontario, Imaging Research Laboratories, Robarts Research Institute, London, ON (Canada); University of Western Ontario, Department of Medical Biophysics, London, ON (Canada)

    2012-09-15

    The goal of this study was to evaluate the ability of balanced steady state free precession (b-SSFP) magnetic resonance imaging sequence to distinguish between live and lysed iron-labelled cells. Human breast cancer cells were labelled with iron oxide nanoparticles. Cells were lysed using sonication. Imaging was performed at 3 T. The timing parameters for b-SSFP and the number of iron-labelled cells in samples were varied to optimise the b-SSFP signal difference between live and lysed iron-labelled cell samples. For in vivo experiments, cells were mixed with Matrigel and implanted into nude mice. Three mice implanted with live labelled cancer cells were irradiated to validate this method. Lysed iron-labelled cells have a significantly higher signal compared with live, intact iron-labelled cells in bSSFP images. The contrast between live and dead cells can be maximised by careful optimisation of timing parameters. A change in the b-SSFP signal was measured 6 days after irradiation, reflecting cell death in vivo. Histology confirmed the presence of dead cells in the implant. Our results show that the b-SSFP sequence can be optimised to allow for the discrimination of live iron-labelled cells and lysed iron-labelled cells in vitro and in vivo. (orig.)

  14. 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}.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Fate in intermittent claudication

    DEFF Research Database (Denmark)

    Jelnes, Rolf; Gaardsting, O; Hougaard Jensen, K

    1986-01-01

    , or an ankle/arm pressure index below 50% were individually significantly associated with progression of the arteriosclerotic disease. These findings show the importance of peripheral blood pressure measurements in the management of patients with intermittent claudication due to arteriosclerotic disease........ The rate of clinical progression of the arteriosclerotic disease (that is, rest pain or gangrene) of the worst affected leg was 7.5% in the first year after referral. Thereafter the rate was 2.2% a year. An ankle systolic blood pressure below 70 mm Hg, a toe systolic blood pressure below 40 mm Hg...... 113 of the patients (44%) had died. Causes of death were no different from those in the general population. Mortality was twice that of the general population matched for age and sex. Mortality among the men was twice that among the women. In men under 60 mortality was four times that expected...

  1. 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

  2. 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

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

    Directory of Open Access Journals (Sweden)

    Qi Tang

    2016-04-01

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

  4. 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.

  5. Multifocus confocal Raman microspectroscopy for fast multimode vibrational imaging of living cells.

    Science.gov (United States)

    Okuno, Masanari; Hamaguchi, Hiro-o

    2010-12-15

    We have developed a multifocus confocal Raman microspectroscopic system for the fast multimode vibrational imaging of living cells. It consists of an inverted microscope equipped with a microlens array, a pinhole array, a fiber bundle, and a multichannel Raman spectrometer. Forty-eight Raman spectra from 48 foci under the microscope are simultaneously obtained by using multifocus excitation and image-compression techniques. The multifocus confocal configuration suppresses the background generated from the cover glass and the cell culturing medium so that high-contrast images are obtainable with a short accumulation time. The system enables us to obtain multimode (10 different vibrational modes) vibrational images of living cells in tens of seconds with only 1 mW laser power at one focal point. This image acquisition time is more than 10 times faster than that in conventional single-focus Raman microspectroscopy.

  6. Live-cell imaging of conidial anastomosis tube fusion during colony initiation in Fusarium oxysporum.

    Directory of Open Access Journals (Sweden)

    Smija M Kurian

    Full Text Available Fusarium oxysporum exhibits conidial anastomosis tube (CAT fusion during colony initiation to form networks of conidial germlings. Here we determined the optimal culture conditions for this fungus to undergo CAT fusion between microconidia in liquid medium. Extensive high resolution, confocal live-cell imaging was performed to characterise the different stages of CAT fusion, using genetically encoded fluorescent labelling and vital fluorescent organelle stains. CAT homing and fusion were found to be dependent on adhesion to the surface, in contrast to germ tube development which occurs in the absence of adhesion. Staining with fluorescently labelled concanavalin A indicated that the cell wall composition of CATs differs from that of microconidia and germ tubes. The movement of nuclei, mitochondria, vacuoles and lipid droplets through fused germlings was observed by live-cell imaging.

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

    Science.gov (United States)

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

    2014-01-15

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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  13. 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.

  14. 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.

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

    Science.gov (United States)

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

    2013-01-01

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

  16. Short-lived, transitory cell-cell interactions foster migration-dependent aggregation.

    Directory of Open Access Journals (Sweden)

    Melissa D Pope

    Full Text Available During embryonic development, motile cells aggregate into cohesive groups, which give rise to tissues and organs. The role of cell migration in regulating aggregation is unclear. The current paradigm for aggregation is based on an equilibrium model of differential cell adhesivity to neighboring cells versus the underlying substratum. In many biological contexts, however, dynamics is critical. Here, we provide evidence that multicellular aggregation dynamics involves both local adhesive interactions and transport by cell migration. Using time-lapse video microscopy, we quantified the duration of cell-cell contacts among migrating cells that collided and adhered to another cell. This lifetime of cell-cell interactions exhibited a monotonic decreasing dependence on substratum adhesivity. Parallel quantitative measurements of cell migration speed revealed that across the tested range of adhesive substrata, the mean time needed for cells to migrate and encounter another cell was greater than the mean adhesion lifetime, suggesting that aggregation dynamics may depend on cell motility instead of the local differential adhesivity of cells. Consistent with this hypothesis, aggregate size exhibited a biphasic dependence on substratum adhesivity, matching the trend we observed for cell migration speed. Our findings suggest a new role for cell motility, alongside differential adhesion, in regulating developmental aggregation events and motivate new design principles for tuning aggregation dynamics in tissue engineering applications.

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. Use of luciferase probes to measure ATP in living cells and animals.

    Science.gov (United States)

    Morciano, Giampaolo; Sarti, Alba Clara; Marchi, Saverio; Missiroli, Sonia; Falzoni, Simonetta; Raffaghello, Lizzia; Pistoia, Vito; Giorgi, Carlotta; Di Virgilio, Francesco; Pinton, Paolo

    2017-08-01

    ATP, the energy exchange factor that connects anabolism and catabolism, is required for major reactions and processes that occur in living cells, such as muscle contraction, phosphorylation and active transport. ATP is also the key molecule in extracellular purinergic signaling mechanisms, with an established crucial role in inflammation and several additional disease conditions. Here, we describe detailed protocols to measure the ATP concentration in isolated living cells and animals using luminescence techniques based on targeted luciferase probes. In the presence of magnesium, oxygen and ATP, the protein luciferase catalyzes oxidation of the substrate luciferin, which is associated with light emission. Recombinantly expressed wild-type luciferase is exclusively cytosolic; however, adding specific targeting sequences can modify its cellular localization. Using this strategy, we have constructed luciferase chimeras targeted to the mitochondrial matrix and the outer surface of the plasma membrane. Here, we describe optimized protocols for monitoring ATP concentrations in the cytosol, mitochondrial matrix and pericellular space in living cells via an overall procedure that requires an average of 3 d. In addition, we present a detailed protocol for the in vivo detection of extracellular ATP in mice using luciferase-transfected reporter cells. This latter procedure may require up to 25 d to complete.

  3. Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

    Science.gov (United States)

    Winckler, Pascale; Lartigue, Lydia; Giannone, Gregory; de Giorgi, Francesca; Ichas, François; Sibarita, Jean-Baptiste; Lounis, Brahim; Cognet, Laurent

    2013-08-01

    Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.

  4. 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.

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

    Science.gov (United States)

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

    2015-02-01

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

  6. 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.

  7. 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)

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  9. 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.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    2017-08-01

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

  12. 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)

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

    Directory of Open Access Journals (Sweden)

    Cervantes Serena

    2012-01-01

    Full Text Available Abstract Background 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. Methods 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. Results 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. Conclusion 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.

  14. 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.

  15. Bioanalytical and chemical sensors using living taste, olfactory, and neural cells and tissues: a short review.

    Science.gov (United States)

    Wu, Chunsheng; Lillehoj, Peter B; Wang, Ping

    2015-11-07

    Biosensors utilizing living tissues and cells have recently gained significant attention as functional devices for chemical sensing and biochemical analysis. These devices integrate biological components (i.e. single cells, cell networks, tissues) with micro-electro-mechanical systems (MEMS)-based sensors and transducers. Various types of cells and tissues derived from natural and bioengineered sources have been used as recognition and sensing elements, which are generally characterized by high sensitivity and specificity. This review summarizes the state of the art in tissue- and cell-based biosensing platforms with an emphasis on those using taste, olfactory, and neural cells and tissues. Many of these devices employ unique integration strategies and sensing schemes based on sensitive transducers including microelectrode arrays (MEAs), field effect transistors (FETs), and light-addressable potentiometric sensors (LAPSs). Several groups have coupled these hybrid biosensors with microfluidics which offers added benefits of small sample volumes and enhanced automation. While this technology is currently limited to lab settings due to the limited stability of living biological components, further research to enhance their robustness will enable these devices to be employed in field and clinical settings.

  16. Deciphering the internal complexity of living cells with quantitative phase microscopy: a multiscale approach

    Science.gov (United States)

    Martinez-Torres, Cristina; Laperrousaz, Bastien; Berguiga, Lotfi; Boyer-Provera, Elise; Elezgaray, Juan; Nicolini, Franck E.; Maguer-Satta, Veronique; Arneodo, Alain; Argoul, Françoise

    2015-09-01

    The distribution of refractive indices (RIs) of a living cell contributes in a nonintuitive manner to its optical phase image and quite rarely can be inverted to recover its internal structure. The interpretation of the quantitative phase images of living cells remains a difficult task because (1) we still have very little knowledge on the impact of its internal macromolecular complexes on the local RI and (2) phase changes produced by light propagation through the sample are mixed with diffraction effects by the internal cell bodies. We propose to implement a two-dimensional wavelet-based contour chain detection method to distinguish internal boundaries based on their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are the morphological indicators suited for comparing cells of different origins and/or to follow their transformation in pathologic situations. We use this method to compare nonadherent blood cells from primary and laboratory culture origins and to assess the internal transformation of hematopoietic stem cells by the transduction of the BCR-ABL oncogene responsible for the chronic myelogenous leukemia.

  17. Naftidrofuryl for intermittent claudication.

    Science.gov (United States)

    De Backer, T L M; Vander Stichele, R; Lehert, P; Van Bortel, L

    2008-04-16

    Lifestyle changes and cardiovascular prevention measures are a primary treatment for intermittent claudication (IC). Symptomatic treatment with vasoactive agents (Anatomic Therapeutic Chemical Classification (ATC) for medicines from the World Health Organisation class CO4A) is controversial. To evaluate evidence on the efficacy and safety of oral naftidrofuryl (ATC CO4 21) versus placebo on the pain-free walking distance (PFWD) of people with IC by using a meta-analysis based on individual patient data (IPD). The Cochrane Peripheral Vascular Diseases Group searched their Trials Register (last searched December 2007) and CENTRAL (last searched 2007, Issue 4). We searched MEDLINE, EMBASE, International Pharmaceutical Abstracts, the Science Citation Index and contacted the authors and checked the reference lists of retrieved articles. We asked the manufacturing company for IPD. We included only randomized controlled trials (RCTs) with low or moderate risk of bias for which the IPD were available. We collected data from the electronic data file or from the case report form and checked the data by a statistical quality control procedure. All randomized patients were analyzed following the intention-to-treat (ITT) principle. The geometric mean of the relative improvement in PFWD was calculated for both treatment groups in all identified studies. The effect of the drug was assessed compared with placebo on final walking distance (WDf) using multilevel and random-effect models and adjusting for baseline walking distance (WD0). For the responder analysis, therapeutic success was defined as an improvement of walking distance of at least 50%. We included seven studies in the IPD (n = 1266 patients). One of these studies (n = 183) was only used in the sensitivity analysis so that the main analysis included 1083 patients. The ratio of the relative improvement in PFWD (naftidrofuryl compared with placebo) was 1.37 (95% confidence interval (CI) 1.32 to 1.51, P < 0.001). The

  18. Three dimensional live-cell STED microscopy at increased depth using a water immersion objective

    Science.gov (United States)

    Heine, Jörn; Wurm, Christian A.; Keller-Findeisen, Jan; Schönle, Andreas; Harke, Benjamin; Reuss, Matthias; Winter, Franziska R.; Donnert, Gerald

    2018-05-01

    Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope's resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5-180 μm.

  19. What is the impact of giant cell arteritis on patients’ lives? A UK qualitative study

    Science.gov (United States)

    Liddle, Jennifer; Bartlam, Roisin; Mallen, Christian D; Mackie, Sarah L; Prior, James A; Helliwell, Toby; Richardson, Jane C

    2017-01-01

    Objectives Clinical management of giant cell arteritis (GCA) involves balancing the risks and burdens arising from the disease with those arising from treatment, but there is little research on the nature of those burdens. We aimed to explore the impact of giant cell arteritis (GCA) and its treatment on patients’ lives. Methods UK patients with GCA participated in semi-structured telephone interviews. Inductive thematic analysis was employed. Results 24 participants were recruited (age: 65–92 years, time since diagnosis: 2 months to >6 years). The overarching themes from analysis were: ongoing symptoms of the disease and its treatment; and ‘life-changing’ impacts. The overall impact of GCA on patients’ lives arose from a changing combination of symptoms, side effects, adaptations to everyday life and impacts on sense of normality. Important factors contributing to loss of normality were glucocorticoid-related treatment burdens and fear about possible future loss of vision. Conclusions The impact of GCA in patients’ everyday lives can be substantial, multifaceted and ongoing despite apparent control of disease activity. The findings of this study will help doctors better understand patient priorities, legitimise patients’ experiences of GCA and work with patients to set realistic treatment goals and plan adaptations to their everyday lives. PMID:28838902

  20. Determination of Peroxisomal pH in Living Mammalian Cells Using pHRed.

    Science.gov (United States)

    Godinho, Luis F; Schrader, Michael

    2017-01-01

    Organelle pH homeostasis is crucial for maintaining proper cellular function. The nature of the peroxisomal pH remains somewhat controversial, with several studies reporting conflicting results. Here, we describe in detail a rapid and accurate method for the measurement of peroxisomal pH, using the pHRed sensor protein and confocal microscopy of living mammalian cells. pHRed, a ratiometric sensor of pH, is targeted to the peroxisomes by virtue of a C-terminal targeting sequence. The probe has a maximum fluorescence emission at 610 nm while exhibiting dual excitation peaks at 440 and 585 nm, allowing for ratiometric imaging and determination of intracellular pH in live cell microscopy.

  1. Conjugated Polymer with Intrinsic Alkyne Units for Synergistically Enhanced Raman Imaging in Living Cells.

    Science.gov (United States)

    Li, Shengliang; Chen, Tao; Wang, Yunxia; Liu, Libing; Lv, Fengting; Li, Zhiliang; Huang, Yanyi; Schanze, Kirk S; Wang, Shu

    2017-10-16

    Development of Raman-active materials with enhanced and distinctive Raman vibrations in the Raman-silent region (1800-2800 cm -1 ) is highly required for specific molecular imaging of living cells with high spatial resolution. Herein, water-soluble cationic conjugated polymers (CCPs), poly(phenylene ethynylene) (PPE) derivatives, are explored for use as alkyne-state-dependent Raman probes for living cell imaging due to synergetic enhancement effect of alkyne vibrations in Raman-silent region compared to alkyne-containing small molecules. The enhanced alkyne signals result from the integration of alkyne groups into the rigid backbone and the delocalized π-conjugated structure. PPE-based conjugated polymer nanoparticles (CPNs) were also prepared as Raman-responsive nanomaterials for distinct imaging application. This work opens a new way into the development of conjugated polymer materials for enhanced Raman imaging. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-06-15

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

  4. A Highly Specific Gold Nanoprobe for Live-Cell Single-Molecule Imaging

    Science.gov (United States)

    Leduc, Cécile; Si, Satyabrata; Gautier, Jérémie; Soto-Ribeiro, Martinho; Wehrle-Haller, Bernhard; Gautreau, Alexis; Giannone, Grégory; Cognet, Laurent; Lounis, Brahim

    2013-04-01

    Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Due to these issues, long-term tracking of proteins in confined and crowded environments, such as intracellular spaces, remains challenging. We have developed a novel optical probe consisting of 5-nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used GFPs with a very high affinity, which we call GFP-nanobodies. These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time. Surface and intracellular GFP-proteins were effectively labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. These nanobody-coated gold nanoparticles are probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.

  5. 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

  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. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-01-15

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

  9. Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing.

    Science.gov (United States)

    Moo, Eng Kuan; Abusara, Ziad; Abu Osman, Noor Azuan; Pingguan-Murphy, Belinda; Herzog, Walter

    2013-08-09

    Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in

  10. Intermedia and Intermittency

    Directory of Open Access Journals (Sweden)

    Veres Bálint

    2014-12-01

    Full Text Available It is commonly known that medial reflections have been initiated by attempts to secure the borders of discrete medial forms and to define the modus operandi of each essentialized medial area. Later on, the focus of study has shifted to plurimedial formations and the interactions between predefined medial genres. In the last few decades, taxonomic approaches to various multi-, inter-, and transmedial phenomena dominated the discussions, which offered invaluable support in mapping the terrain, but at the same time hindered the analysis of the ephemeral, time-dependent aspects of plurimedial operations. While we explore the properties of each medial configuration, we lose sight of the actual historical drivers that produce ever-new configurations. My thesis is that any discourse on intermediality should be paralleled by a discourse on cultural intermittency, and consequently, media studies should involve an approach that focuses on the “ecosystem” of the constantly renewing media configurations from the point of view of their vitalizing potential and capability to trigger heightened experiences. This approach draws much inspiration from K. Ludwig Pfeiffer’s media anthropology that gives orientation in my paper.

  11. 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

  12. 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.

  13. Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms

    Science.gov (United States)

    Sipieter, François; Cappe, Benjamin; Gonzalez Pisfil, Mariano; Spriet, Corentin; Bodart, Jean-François; Cailliau-Maggio, Katia; Vandenabeele, Peter; Héliot, Laurent; Riquet, Franck B.

    2015-01-01

    Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues. PMID:26517832

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

    Science.gov (United States)

    Montoya, Leticia A; Pluth, Michael D

    2012-05-16

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

  15. 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.)

  16. Chronic Intermittent Hypoxia Induces Atherosclerosis

    OpenAIRE

    Savransky, Vladimir; Nanayakkara, Ashika; Li, Jianguo; Bevans, Shannon; Smith, Philip L.; Rodriguez, Annabelle; Polotsky, Vsevolod Y.

    2007-01-01

    Rationale: Obstructive sleep apnea, a condition leading to chronic intermittent hypoxia (CIH), is associated with hyperlipidemia, atherosclerosis, and a high cardiovascular risk. A causal link between obstructive sleep apnea and atherosclerosis has not been established.

  17. 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

  18. Direct Light-up of cAMP Derivatives in Living Cells by Click Reactions

    Directory of Open Access Journals (Sweden)

    Yan Xu

    2013-10-01

    Full Text Available 8-Azidoadenosine 3′,5′-cyclic monophosphate (8-azido cAMP was directly detected in living cells, by applying Cu-free azide-alkyne cycloaddition to probe cAMP derivatives by fluorescence light-up. Fluorescence emission was generated by two non-fluorescent molecules, 8-azido cAMP as a model target and difluorinated cyclooctyne (DIFO reagent as a probe. The azide-alkyne cycloaddition reaction between 8-azido cAMP and DIFO induces fluorescence in 8-azido cAMP. The fluorescence emission serves as a way to probe 8-azido cAMP in cells.

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

    Science.gov (United States)

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

    2018-06-01

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

  20. Inhibition of Reporter Genes by Small Interfering RNAs in Cell Culture and Living Fish

    DEFF Research Database (Denmark)

    Larashati, Sekar; Schyth, Brian Dall; Lorenzen, Niels

    2011-01-01

    be used to observe the knock down effect by siRNAs designed to target these reporters. One aim of this project is to verify the specific knock down effect of siRNAs in cell culture and in living fish and to establish easy-read out models for testing the effect especially in vivo. Cell culture from human...... coinjection and the assay is important in order to detect knock down by siRNA. Our experiment reveal in vivo knock down at 72 hours post injection of reporter gene and siRNA, but further dose-response experiments are required to confirm specifity....

  1. The lived experience of autologous stem cell-transplanted patients: Post-transplantation and before discharge.

    Science.gov (United States)

    Alnasser, Qasem; Abu Kharmah, Salahel Deen; Attia, Manal; Aljafari, Akram; Agyekum, Felicia; Ahmed, Falak Aftab

    2018-04-01

    To explore the lived experience of the patients post-haematopoietic stem cell transplantation and specifically after engraftment and before discharge. Patients post-stem cell transplantation experience significant changes in all life aspects. Previous studies carried out by other researchers focused mainly on the postdischarge experience, where patients reported their perceptions that have always been affected by the life post-transplantation and influenced by their surroundings. The lived experience of patients, specifically after engraftment and prior to discharge (the "transition" phase), has not been adequately explored in the literature. Doing so might provide greater insight into the cause of change post-haematopoietic stem cell transplantation. This study is a phenomenological description of the participants' perception about their lived experience post-haematopoietic stem cell transplantation. The study used Giorgi's method of analysis. Through purposive sampling, 15 post-haematopoietic stem cell transplantation patients were recruited. Data were collected by individual interviews. Data were then analysed based on Giorgi's method of analysis to reveal the meaning of a phenomenon as experienced through the identification of essential themes. The analysis process revealed 12 core themes covered by four categories that detailed patients lived experience post-haematopoietic stem cell transplantation. The four categories were general transplant experience, effects of transplantation, factors of stress alleviation and finally life post-transplantation. This study showed how the haematopoietic stem cell transplantation affected the patients' physical, psychological and spiritual well-being. Transplantation also impacted on the patients' way of thinking and perception of life. Attending to patients' needs during transplantation might help to alleviate the severity of the effects and therefore improve experience. Comprehensive information about transplantation needs

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

    Directory of Open Access Journals (Sweden)

    Adam D Hoppe

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

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

    KAUST Repository

    Dreissig, Steven

    2017-05-16

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

  4. Dynamic characterizers of spatiotemporal intermittency

    OpenAIRE

    Gupte, Neelima; Jabeen, Zahera

    2006-01-01

    Systems of coupled sine circle maps show regimes of spatiotemporally intermittent behaviour with associated scaling exponents which belong to the DP class, as well as regimes of spatially intermittent behaviour (with associated regular dynamical behaviour) which do not belong to the DP class. Both types of behaviour are seen along the bifurcation boundaries of the synchronized solutions, and contribute distinct signatures to the dynamical characterizers of the system, viz. the distribution of...

  5. Real-time monitoring of caspase cascade activation in living cells.

    Science.gov (United States)

    Zhu, Lei; Huang, Xinglu; Choi, Ki Young; Ma, Ying; Zhang, Fan; Liu, Gang; Lee, Seulki; Chen, Xiaoyuan

    2012-10-10

    We introduce a simple, versatile and robust one-step technique that enables real-time imaging of multiple intracellular caspase activities in living cells without the need for complicated synthetic protocols. Conventional fluorogenic probes or recently reported activatable probes have been designed to target various proteases but are limited to extracellular molecules. Only a few have been applied to image intracellular proteases in living cells because most of these probes have limited cell-permeability. Our platform does not need complicated synthetic processes; instead it involves a straightforward peptide synthesis and a simple mixing step with a commercial transfection agent. The transfection agent efficiently delivered the highly quenched fluorogenic probes, comprised of distinctive pairs of dyes and quenchers, to the initiator caspase-8 and the effector caspase-3 in MDA-MB-435 cells, allowing dual-imaging of the activities of both caspases during the apoptotic process induced by TNF-related apoptosis induced ligand (TRAIL). With the combination of multiple fluorogenic probes, this simple platform can be applied to multiplexed imaging of selected intracellular proteases to study apoptotic processes in pathologies or for cell-based high throughput screening systems for drug discovery. Published by Elsevier B.V.

  6. Open-dish incubator for live cell imaging with an inverted microscope.

    Science.gov (United States)

    Heidemann, Steven R; Lamoureux, Phillip; Ngo, Kha; Reynolds, Matthew; Buxbaum, Robert E

    2003-10-01

    Here we describe the design and fabrication of an inexpensive cell culture incubator for the stage of an inverted light microscope for use in live cell imaging. This device maintains the temperature of the cell culture at 37 degrees C with great stability and, after reaching equilibrium, provides focal stability of an image for 20-25 min with oil-immersion lenses. We describe two versions of the incubator: one for use with standard 60-mm plastic culture dishes, and the other version for imaging of cells on glass coverslips. Either can be made for less than $400. Most components are widely available commercially, and it requires only simple wiring and 3 h to assemble. Although the device is generally useful for live cell imaging on an inverted microscope, it is particularly suitable for work in which instruments are introduced into the culture, such as electrophysiology or micromanipulation. The design is based on the principle that control performance is limited by the lag time between detection and response. The key element of the design is a heated, temperature-controlled aluminum ring serving as a mini-incubator surrounding the culture vessel. For this reason, we call our design a "ringcubator."

  7. Study of metal bioaccumulation by nuclear microprobe analysis of algae fossils and living algae cells

    International Nuclear Information System (INIS)

    Guo, P.; Wang, J.; Li, X.; Zhu, J.; Reinert, T.; Heitmann, J.; Spemann, D.; Vogt, J.; Flagmeyer, R.-H.; Butz, T.

    2000-01-01

    Microscopic ion-beam analysis of palaeo-algae fossils and living green algae cells have been performed to study the metal bioaccumulation processes. The algae fossils, both single cellular and multicellular, are from the late Neoproterozonic (570 million years ago) ocean and perfectly preserved within a phosphorite formation. The biosorption of the rare earth element ions Nd 3+ by the green algae species euglena gracilis was investigated with a comparison between the normal cells and immobilized ones. The new Leipzig Nanoprobe, LIPSION, was used to produce a proton beam with 2 μm size and 0.5 nA beam current for this study. PIXE and RBS techniques were used for analysis and imaging. The observation of small metal rich spores (<10 μm) surrounding both of the fossils and the living cells proved the existence of some specific receptor sites which bind metal carrier ligands at the microbic surface. The bioaccumulation efficiency of neodymium by the algae cells was 10 times higher for immobilized algae cells. It confirms the fact that the algae immobilization is an useful technique to improve its metal bioaccumulation

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  9. Living biointerfaces based on non-pathogenic bacteria support stem cell differentiation

    Science.gov (United States)

    Hay, Jake J.; Rodrigo-Navarro, Aleixandre; Hassi, Karoliina; Moulisova, Vladimira; Dalby, Matthew J.; Salmeron-Sanchez, Manuel

    2016-02-01

    Lactococcus lactis, a non-pathogenic bacteria, has been genetically engineered to express the III7-10 fragment of human fibronectin as a membrane protein. The engineered L. lactis is able to develop biofilms on different surfaces (such as glass and synthetic polymers) and serves as a long-term substrate for mammalian cell culture, specifically human mesenchymal stem cells (hMSC). This system constitutes a living interface between biomaterials and stem cells. The engineered biofilms remain stable and viable for up to 28 days while the expressed fibronectin fragment induces hMSC adhesion. We have optimised conditions to allow long-term mammalian cell culture, and found that the biofilm is functionally equivalent to a fibronectin-coated surface in terms of osteoblastic differentiation using bone morphogenetic protein 2 (BMP-2) added to the medium. This living bacteria interface holds promise as a dynamic substrate for stem cell differentiation that can be further engineered to express other biochemical cues to control hMSC differentiation.

  10. A Checklist for Successful Quantitative Live Cell Imaging in Systems Biology

    Science.gov (United States)

    Sung, Myong-Hee

    2013-01-01

    Mathematical modeling of signaling and gene regulatory networks has provided unique insights about systems behaviors for many cell biological problems of medical importance. Quantitative single cell monitoring has a crucial role in advancing systems modeling of molecular networks. However, due to the multidisciplinary techniques that are necessary for adaptation of such systems biology approaches, dissemination to a wide research community has been relatively slow. In this essay, I focus on some technical aspects that are often under-appreciated, yet critical in harnessing live cell imaging methods to achieve single-cell-level understanding and quantitative modeling of molecular networks. The importance of these technical considerations will be elaborated with examples of successes and shortcomings. Future efforts will benefit by avoiding some pitfalls and by utilizing the lessons collectively learned from recent applications of imaging in systems biology. PMID:24709701

  11. Live imaging of spindle pole disorganization in docetaxel-treated multicolor cells

    International Nuclear Information System (INIS)

    Sakaushi, Shinji; Nishida, Kumi; Minamikawa, Harumi; Fukada, Takashi; Oka, Shigenori; Sugimoto, Kenji

    2007-01-01

    Treatment of cells with docetaxel at low concentrations induces aberrant bipolar spindles of which two centrosomes stay at only one pole, and also induces multipolar spindles. To gain insight into the relations between centrosome impairment and structural defects of the spindle, live-cell imaging was performed on a human MDA Auro/imp/H3 cell line in which centrosomes/mitotic spindles, nuclear membrane and chromatin were simultaneously visualized by fluorescent proteins. In the presence of docetaxel at IC 50 concentration, the centrosomes did not segregate, and multiple aster-like structures ectopically arose around the disappearing nuclear membrane. Those ectopic structures formed an acentrosomal pole opposing to the two-centrosomes-containing pole. In late metaphase, one pole often fragmented into multiple spindle poles, leading multipolar division. These results suggest that spindle pole fragility may be induced by centrosome impairment, and collapse of the pole may contribute to induction of aneuploid daughter cells

  12. New Insights into HTLV-1 Particle Structure, Assembly, and Gag-Gag Interactions in Living Cells

    Directory of Open Access Journals (Sweden)

    Jolene L. Johnson

    2011-06-01

    Full Text Available Human T-cell leukemia virus type 1 (HTLV-1 has a reputation for being extremely difficult to study in cell culture. The challenges in propagating HTLV-1 has prevented a rigorous analysis of how these viruses replicate in cells, including the detailed steps involved in virus assembly. The details for how retrovirus particle assembly occurs are poorly understood, even for other more tractable retroviral systems. Recent studies on HTLV-1 using state-of-the-art cryo-electron microscopy and fluorescence-based biophysical approaches explored questions related to HTLV-1 particle size, Gag stoichiometry in virions, and Gag-Gag interactions in living cells. These results provided new and exciting insights into fundamental aspects of HTLV-1 particle assembly—which are distinct from those of other retroviruses, including HIV-1. The application of these and other novel biophysical approaches promise to provide exciting new insights into HTLV-1 replication.

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

    Science.gov (United States)

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

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

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

    Science.gov (United States)

    Wong, Andrew D; Searson, Peter C

    2014-09-01

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

  15. High-speed force mapping on living cells with a small cantilever atomic force microscope

    International Nuclear Information System (INIS)

    Braunsmann, Christoph; Seifert, Jan; Rheinlaender, Johannes; Schäffer, Tilman E.

    2014-01-01

    The imaging speed of the wide-spread force mapping mode for quantitative mechanical measurements on soft samples in liquid with the atomic force microscope (AFM) is limited by the bandwidth of the z-scanner and viscous drag forces on the cantilever. Here, we applied high-speed, large scan-range atomic force microscopy and small cantilevers to increase the speed of force mapping by ≈10−100 times. This allowed resolving dynamic processes on living mouse embryonic fibroblasts. Cytoskeleton reorganization during cell locomotion, growth of individual cytoskeleton fibers, cell blebbing, and the formation of endocytic pits in the cell membrane were observed. Increasing the force curve rate from 2 to 300 Hz increased the measured apparent Young's modulus of the cells by about 10 times, which facilitated force mapping measurements at high speed

  16. High-speed force mapping on living cells with a small cantilever atomic force microscope

    Energy Technology Data Exchange (ETDEWEB)

    Braunsmann, Christoph; Seifert, Jan; Rheinlaender, Johannes; Schäffer, Tilman E., E-mail: Tilman.Schaeffer@uni-tuebingen [Institute of Applied Physics and LISA, University of Tübingen, Auf der Morgenstelle 10, 72076 Tübingen (Germany)

    2014-07-15

    The imaging speed of the wide-spread force mapping mode for quantitative mechanical measurements on soft samples in liquid with the atomic force microscope (AFM) is limited by the bandwidth of the z-scanner and viscous drag forces on the cantilever. Here, we applied high-speed, large scan-range atomic force microscopy and small cantilevers to increase the speed of force mapping by ≈10−100 times. This allowed resolving dynamic processes on living mouse embryonic fibroblasts. Cytoskeleton reorganization during cell locomotion, growth of individual cytoskeleton fibers, cell blebbing, and the formation of endocytic pits in the cell membrane were observed. Increasing the force curve rate from 2 to 300 Hz increased the measured apparent Young's modulus of the cells by about 10 times, which facilitated force mapping measurements at high speed.

  17. Delivery of Optical Contrast Agents using Triton-X100, Part 1: Reversible permeabilization of live cells for intracellular labeling

    OpenAIRE

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

    2009-01-01

    Effective delivery of optical contrast agents into live cells remains a significant challenge. We sought to determine whether Triton-X100, a detergent commonly used for membrane isolation and protein purification, could be used to effectively and reversibly permeabilize live cells for delivery of targeted optical contrast agents. Although Triton-X100 is widely recognized as a good cell permeabilization agent, no systematic study has evaluated the efficiency, reproducibility, and reversibility...

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

    Science.gov (United States)

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

    2018-05-08

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Xiaoliang Sunney Xie

    2009-03-30

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

  20. Mathematical imaging methods for mitosis analysis in live-cell phase contrast microscopy.

    Science.gov (United States)

    Grah, Joana Sarah; Harrington, Jennifer Alison; Koh, Siang Boon; Pike, Jeremy Andrew; Schreiner, Alexander; Burger, Martin; Schönlieb, Carola-Bibiane; Reichelt, Stefanie

    2017-02-15

    In this paper we propose a workflow to detect and track mitotic cells in time-lapse microscopy image sequences. In order to avoid the requirement for cell lines expressing fluorescent markers and the associated phototoxicity, phase contrast microscopy is often preferred over fluorescence microscopy in live-cell imaging. However, common specific image characteristics complicate image processing and impede use of standard methods. Nevertheless, automated analysis is desirable due to manual analysis being subjective, biased and extremely time-consuming for large data sets. Here, we present the following workflow based on mathematical imaging methods. In the first step, mitosis detection is performed by means of the circular Hough transform. The obtained circular contour subsequently serves as an initialisation for the tracking algorithm based on variational methods. It is sub-divided into two parts: in order to determine the beginning of the whole mitosis cycle, a backwards tracking procedure is performed. After that, the cell is tracked forwards in time until the end of mitosis. As a result, the average of mitosis duration and ratios of different cell fates (cell death, no division, division into two or more daughter cells) can be measured and statistics on cell morphologies can be obtained. All of the tools are featured in the user-friendly MATLAB®Graphical User Interface MitosisAnalyser. Copyright © 2017. Published by Elsevier Inc.

  1. An improved model for nucleation-limited ice formation in living cells during freezing.

    Directory of Open Access Journals (Sweden)

    Jingru Yi

    Full Text Available Ice formation in living cells is a lethal event during freezing and its characterization is important to the development of optimal protocols for not only cryopreservation but also cryotherapy applications. Although the model for probability of ice formation (PIF in cells developed by Toner et al. has been widely used to predict nucleation-limited intracellular ice formation (IIF, our data of freezing Hela cells suggest that this model could give misleading prediction of PIF when the maximum PIF in cells during freezing is less than 1 (PIF ranges from 0 to 1. We introduce a new model to overcome this problem by incorporating a critical cell volume to modify the Toner's original model. We further reveal that this critical cell volume is dependent on the mechanisms of ice nucleation in cells during freezing, i.e., surface-catalyzed nucleation (SCN and volume-catalyzed nucleation (VCN. Taken together, the improved PIF model may be valuable for better understanding of the mechanisms of ice nucleation in cells during freezing and more accurate prediction of PIF for cryopreservation and cryotherapy applications.

  2. Dissecting Regional Variations in Stress Fiber Mechanics in Living Cells with Laser Nanosurgery

    Energy Technology Data Exchange (ETDEWEB)

    Tanner, Kandice; Boudreau, Aaron; Bissell, Mina J; Kumar, Sanjay

    2010-03-02

    The ability of a cell to distribute contractile stresses across the extracellular matrix in a spatially heterogeneous fashion underlies many cellular behaviors, including motility and tissue assembly. Here we investigate the biophysical basis of this phenomenon by using femtosecond laser nanosurgery to measure the viscoelastic recoil and cell-shape contributions of contractile stress fibers (SFs) located in specific compartments of living cells. Upon photodisruption and recoil, myosin light chain kinase-dependent SFs located along the cell periphery display much lower effective elasticities and higher plateau retraction distances than Rho-associated kinase-dependent SFs located in the cell center, with severing of peripheral fibers uniquely triggering a dramatic contraction of the entire cell within minutes of fiber irradiation. Image correlation spectroscopy reveals that when one population of SFs is pharmacologically dissipated, actin density flows toward the other population. Furthermore, dissipation of peripheral fibers reduces the elasticity and increases the plateau retraction distance of central fibers, and severing central fibers under these conditions triggers cellular contraction. Together, these findings show that SFs regulated by different myosin activators exhibit different mechanical properties and cell shape contributions. They also suggest that some fibers can absorb components and assume mechanical roles of other fibers to stabilize cell shape.

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

    Science.gov (United States)

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

    2015-01-01

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

  4. Evaluation of chemical fluorescent dyes as a protein conjugation partner for live cell imaging.

    Directory of Open Access Journals (Sweden)

    Yoko Hayashi-Takanaka

    Full Text Available To optimize live cell fluorescence imaging, the choice of fluorescent substrate is a critical factor. Although genetically encoded fluorescent proteins have been used widely, chemical fluorescent dyes are still useful when conjugated to proteins or ligands. However, little information is available for the suitability of different fluorescent dyes for live imaging. We here systematically analyzed the property of a number of commercial fluorescent dyes when conjugated with antigen-binding (Fab fragments directed against specific histone modifications, in particular, phosphorylated H3S28 (H3S28ph and acetylated H3K9 (H3K9ac. These Fab fragments were conjugated with a fluorescent dye and loaded into living HeLa cells. H3S28ph-specific Fab fragments were expected to be enriched in condensed chromosomes, as H3S28 is phosphorylated during mitosis. However, the degree of Fab fragment enrichment on mitotic chromosomes varied depending on the conjugated dye. In general, green fluorescent dyes showed higher enrichment, compared to red and far-red fluorescent dyes, even when dye:protein conjugation ratios were similar. These differences are partly explained by an altered affinity of Fab fragment after dye-conjugation; some dyes have less effect on the affinity, while others can affect it more. Moreover, red and far-red fluorescent dyes tended to form aggregates in the cytoplasm. Similar results were observed when H3K9ac-specific Fab fragments were used, suggesting that the properties of each dye affect different Fab fragments similarly. According to our analysis, conjugation with green fluorescent dyes, like Alexa Fluor 488 and Dylight 488, has the least effect on Fab affinity and is the best for live cell imaging, although these dyes are less photostable than red fluorescent dyes. When multicolor imaging is required, we recommend the following dye combinations for optimal results: Alexa Fluor 488 (green, Cy3 (red, and Cy5 or CF640 (far-red.

  5. Engineering Multifunctional Living Paints: Thin, Convectively-Assembled Biocomposite Coatings of Live Cells and Colloidal Latex Particles Deposited by Continuous Convective-Sedimentation Assembly

    Science.gov (United States)

    Jenkins, Jessica Shawn

    Advanced composite materials could be revolutionized by the development of methods to incorporate living cells into functional materials and devices. This could be accomplished by continuously and rapidly depositing thin ordered arrays of adhesive colloidal latex particles and live cells that maintain stability and preserve microbial reactivity. Convective assembly is one method of rapidly assembling colloidal particles into thin (advantages over thicker randomly ordered composites, including enhanced cell stability and increased reactivity through minimized diffusion resistance to nutrients and reduced light scattering. This method can be used to precisely deposit live bacteria, cyanobacteria, yeast, and algae into biocomposite coatings, forming reactive biosensors, photoabsorbers, or advanced biocatalysts. This dissertation developed new continuous deposition and coating characterization methods for fabricating and characterizing 90 hours) photohydrogen production under anoxygenic conditions. Nutrient reduction slows cell division, minimizing coating outgrowth, and promotes photohydrogen generation, improving coating reactivity. Scanning electron microscopy of microstructure revealed how coating reactivity can be controlled by the size and distribution of the nanopores in the biocomposite layers. Variations in colloid microsphere size and suspension composition do not affect coating reactivity, but both parameters alter coating microstructure. Porous paper coated with thin coatings of colloidal particles and cells to enable coatings to be used in a gas-phase without dehydration may offer higher volumetric productivity for hydrogen production. Future work should focus on optimization of cell density, light intensity, media cycling, and acetate concentration.

  6. Evaluation of royal jelly as an alternative to fetal bovine serum in cell culture using cell proliferation assays and live cell imaging.

    Science.gov (United States)

    Musa, Marahaini; Nasir, Nurul Fatihah Mohamad; Thirumulu, Kannan Ponnuraj

    2014-01-01

    Royal jelly is a nutritious substance produced by the young nurse bees and contains significant amounts of proteins which are important for cell growth and proliferation. The aim of this study was to evaluate the effect of royal jelly as an alternative to fetal bovine serum (FBS) in cell culture using cell proliferation assays and live cell imaging. MRC-5 cells were treated with various concentrations of royal jelly extract in MTT assay. The control groups were comprised of Alpha-Minimal Essential Medium (α-MEM) alone and α-MEM with 10% FBS. Subsequently, the cell proliferation was studied for 10 days using Alamar Blue assay and live cell imaging from 48 to 72 h. The population doubling time (PDT) was determined using trypan blue assay after live cell imaging. In MTT assay, 0.156 and 0.078 mg/ml of royal jelly produced higher cell viability compared to positive control group but were not significantly different (P > 0.05). In the Alamar Blue assay, 0.156 and 0.078 mg/ml of royal jelly produced greater percentage of reduction at day 3 even though no significant difference was found (P > 0.05). Based on live cell imaging, the PDT for positive, negative, 0.156 and 0.078 mg/ml of royal jelly groups were 29.09, 62.50, 41.67 and 41.67 h respectively. No significant difference was found in the PDT between all the groups (P > 0.05). Royal jelly does not exhibit similar ability like FBS to facilitate cell growth under the present test conditions.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-10-07

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

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

    Science.gov (United States)

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

    2016-08-10

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

  9. Live celloidosome structures based on the assembly of individual cells by colloid interactions.

    Science.gov (United States)

    Fakhrullin, Rawil F; Brandy, Marie-Laure; Cayre, Olivier J; Velev, Orlin D; Paunov, Vesselin N

    2010-10-14

    A new class of colloid structures, celloidosomes, has been developed which represent hollow microcapsules whose membranes consist of a single monolayer of living cells. Two routes for producing these structures were designed based on templating of: (i) air bubbles and (ii) anisotropic microcrystals of calcium carbonate with living cells, which allowed us to fabricate celloidosomes of spherical, rhombohedral and needle-like morphologies. Air microbubbles were templated by yeast cells coated with poly(allylamine hydrochloride) (PAH), then coated with carboxymethylcellulose and rehydrated resulting in the formation of spherical multicellular structures. Similarly, calcium carbonate microcrystals of anisotropic shapes were coated with several consecutive layers of oppositely charged polyelectrolytes to obtain a positive surface charge which was used to immobilise yeast cells coated with anionic polyelectrolyte of their surfaces. After dissolving of sacrificial cores, hollow multicellular structures were obtained. The viability of the cells in the produced structures was confirmed by using fluorescein diacetate. In order to optimize the separation of celloidosomes from free cells magnetic nanoparticles were immobilised onto the surface of templates prior to the cells deposition, which greatly facilitated the separation using a permanent magnet. Two alternative approaches were developed to form celloidosome structures using magnetically functionalised core-shell microparticles which resulted in the formation of celloidosomes with needle-like and cubic-like geometries which follows the original morphology of the calcium carbonate microcrystals. Our methods for fabrication of celloidosomes may found applications in the development of novel symbiotic bio-structures, artificial multicellular organisms and in tissue engineering. The unusual structure of celloidosomes resembles the primitive forms of multicellular species, like Volvox, and other algae and could be regarded as

  10. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

    Directory of Open Access Journals (Sweden)

    Wiltrud Haaß

    Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

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

    Science.gov (United States)

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

    2014-12-01

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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

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

  13. Intermittent Fasting Modulation of the Diabetic Syndrome in Streptozotocin-Injected Rats

    Directory of Open Access Journals (Sweden)

    Louiza Belkacemi

    2012-01-01

    Full Text Available This study investigates the effects of intermittent overnight fasting in streptozotocin-induced diabetic rats (STZ rats. Over 30 days, groups of 5-6 control or STZ rats were allowed free food access, starved overnight, or exposed to a restricted food supply comparable to that ingested by the intermittently fasting animals. Intermittent fasting improved glucose tolerance, increased plasma insulin, and lowered Homeostatis Model Assessment index. Caloric restriction failed to cause such beneficial effects. The β-cell mass, as well as individual β-cell and islet area, was higher in intermittently fasting than in nonfasting STZ rats, whilst the percentage of apoptotic β-cells appeared lower in the former than latter STZ rats. In the calorie-restricted STZ rats, comparable findings were restricted to individual islet area and percentage of apoptotic cells. Hence, it is proposed that intermittent fasting could represent a possible approach to prevent or minimize disturbances of glucose homeostasis in human subjects.

  14. Photoacoustic imaging of mesenchymal stem cells in living mice via silica-coated gold nanorods

    Science.gov (United States)

    Jokerst, Jesse V.; Thangaraj, Mridhula; Gambhir, Sanjiv S.

    2014-03-01

    Imaging is crucial for stem cell therapy to monitor the location(s), numbers, and state of the implanted cells. Real-time imaging in particular can ensure proper cell delivery for best engraftment. However, established imaging tools such as MRI are limited by their temporal resolution for guidance during delivery. In contrast, photoacoustic imaging is ideally suited for real time, image-guided therapy. Here, we use silica-coated gold nanorods as photoacoustic contrast agents and deploy them to image and quantitate mesenchymal stem cells during implant into the muscle tissue of live mice. Silica-coated gold nanorods (SiGNRs) were created with standard methods and loaded into mesenchymal stem cells (MSCs) without transfection agents. There was no significant (pmuscle tissue to simulate a muscular dystrophy patient. Mice (N=5) treated with these SiGNRlabeled MSCs exhibited no adverse events and implants up to 5 mm deep were easily visualized. The in vivo detection limit was 90,000 cells in a 100 uL bolus in mouse thigh muscle. Here, the B-mode signal is useful for orienting the treatment area and visualizing the delivery catheter while the photoacoustic mode offers cell-specific content. The photoacoustic signal was validated with histology a long-term fluorescent tracking dye after MSC transplant.

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

    Science.gov (United States)

    Jing, Chaoran

    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 engineering fluorogenic trimethoprim-based chemical tags (TMP-tags) in which the fluorophore is quenched until binding with E. coli dihydrofolate reductase (eDHFR) tagged protein displaces the quencher. Thus, we began by building a non-fluorogenic, covalent TMP-tag based on a proximity-induced reaction known to achieve rapid and specific labeling both in vitro and inside of living cells. Here we take the final step and render the covalent TMP-tag fluorogenic. In brief, we designed a trimeric TMP-fluorophore-quencher molecule (TMP-Q-Atto520) with the quencher attached to a leaving group that, upon TMP binding to eDHFR, would be cleaved by a cysteine residue (Cys) installed just outside the binding pocket of eDHFR. We present the in vitro experiments showing that the eDHFR:L28C nucleophile cleaves the TMP-Q-Atto520 rapidly and efficiently, resulting in covalent labeling and remarkable fluorescence enhancement. Most significantly, while only our initial design, TMP-Q-Atto520 achieved the demanding goal of not only labeling highly abundant, localized intracellular proteins, but also less abundant, more dynamic cytoplasmic proteins. These results suggest that fluorogenic TMP-tag can significantly impact highresolution live cell imaging and further establish the potential of proximity-induced reactivity and organic chemistry more broadly as part of the growing toolbox for synthetic biology and cell engineering. PMID:23745575

  16. A Molecular Probe for the Detection of Polar Lipids in Live Cells.

    Science.gov (United States)

    Bader, Christie A; Shandala, Tetyana; Carter, Elizabeth A; Ivask, Angela; Guinan, Taryn; Hickey, Shane M; Werrett, Melissa V; Wright, Phillip J; Simpson, Peter V; Stagni, Stefano; Voelcker, Nicolas H; Lay, Peter A; Massi, Massimiliano; Plush, Sally E; Brooks, Douglas A

    2016-01-01

    Lipids have an important role in many aspects of cell biology, including membrane architecture/compartment formation, intracellular traffic, signalling, hormone regulation, inflammation, energy storage and metabolism. Lipid biology is therefore integrally involved in major human diseases, including metabolic disorders, neurodegenerative diseases, obesity, heart disease, immune disorders and cancers, which commonly display altered lipid transport and metabolism. However, the investigation of these important cellular processes has been limited by the availability of specific tools to visualise lipids in live cells. Here we describe the potential for ReZolve-L1™ to localise to intracellular compartments containing polar lipids, such as for example sphingomyelin and phosphatidylethanolamine. In live Drosophila fat body tissue from third instar larvae, ReZolve-L1™ interacted mainly with lipid droplets, including the core region of these organelles. The presence of polar lipids in the core of these lipid droplets was confirmed by Raman mapping and while this was consistent with the distribution of ReZolve-L1™ it did not exclude that the molecular probe might be detecting other lipid species. In response to complete starvation conditions, ReZolve-L1™ was detected mainly in Atg8-GFP autophagic compartments, and showed reduced staining in the lipid droplets of fat body cells. The induction of autophagy by Tor inhibition also increased ReZolve-L1™ detection in autophagic compartments, whereas Atg9 knock down impaired autophagosome formation and altered the distribution of ReZolve-L1™. Finally, during Drosophila metamorphosis fat body tissues showed increased ReZolve-L1™ staining in autophagic compartments at two hours post puparium formation, when compared to earlier developmental time points. We concluded that ReZolve-L1™ is a new live cell imaging tool, which can be used as an imaging reagent for the detection of polar lipids in different intracellular

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

    International Nuclear Information System (INIS)

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

    2010-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-04-09

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

  19. Noninvasive micromanipulation of live HIV-1 infected cells via laser light

    Science.gov (United States)

    Mthunzi, Patience

    2015-12-01

    Live mammalian cells from various tissues of origin can be aseptically and noninvasively micromanipulated via lasers of different regimes. Laser-driven techniques are therefore paving a path toward the advancement of human immuno-deficiency virus (HIV-1) investigations. Studies aimed at the interaction of laser light, nanomaterials, and biological materials can also lead to an understanding of a wealth of disease conditions and result in photonics-based therapies and diagnostic tools. Thus, in our research, both continuous wave and pulsed lasers operated at varying wavelengths are employed, as they possess special properties that allow classical biomedical applications. This paper discusses photo-translocation of antiretroviral drugs into HIV-1 permissive cells and preliminary results of low-level laser therapy (LLLT) in HIV-1 infected cells.

  20. Localized Chemical Remodeling for Live Cell Imaging of Protein-Specific Glycoform.

    Science.gov (United States)

    Hui, Jingjing; Bao, Lei; Li, Siqiao; Zhang, Yi; Feng, Yimei; Ding, Lin; Ju, Huangxian

    2017-07-03

    Live cell imaging of protein-specific glycoforms is important for the elucidation of glycosylation mechanisms and identification of disease states. The currently used metabolic oligosaccharide engineering (MOE) technology permits routinely global chemical remodeling (GCM) for carbohydrate site of interest, but can exert unnecessary whole-cell scale perturbation and generate unpredictable metabolic efficiency issue. A localized chemical remodeling (LCM) strategy for efficient and reliable access to protein-specific glycoform information is reported. The proof-of-concept protocol developed for MUC1-specific terminal galactose/N-acetylgalactosamine (Gal/GalNAc) combines affinity binding, off-on switchable catalytic activity, and proximity catalysis to create a reactive handle for bioorthogonal labeling and imaging. Noteworthy assay features associated with LCM as compared with MOE include minimum target cell perturbation, short reaction timeframe, effectiveness as a molecular ruler, and quantitative analysis capability. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. From Never Born Proteins to Minimal Living Cells: two projects in synthetic biology.

    Science.gov (United States)

    Luisi, Pier Luigi; Chiarabelli, Cristiano; Stano, Pasquale

    2006-12-01

    The Never Born Proteins (NBPs) and the Minimal Cell projects are two currently developed research lines belonging to the field of synthetic biology. The first deals with the investigation of structural and functional properties of de novo proteins with random sequences, selected and isolated using phage display methods. The minimal cell is the simplest cellular construct which displays living properties, such as self-maintenance, self-reproduction and evolvability. The semi-synthetic approach to minimal cells involves the use of extant genes and proteins in order to build a supramolecular construct based on lipid vesicles. Results and outlooks on these two research lines are shortly discussed, mainly focusing on their relevance to the origin of life studies.

  2. Relaxation distribution function of intracellular dielectric zones as an indicator of tumorous transition of living cells.

    Science.gov (United States)

    Thornton, B S; Hung, W T; Irving, J

    1991-01-01

    The response decay data of living cells subject to electric polarization is associated with their relaxation distribution function (RDF) and can be determined using the inverse Laplace transform method. A new polynomial, involving a series of associated Laguerre polynomials, has been used as the approximating function for evaluating the RDF, with the advantage of avoiding the usual arbitrary trial values of a particular parameter in the numerical computations. Some numerical examples are given, followed by an application to cervical tissue. It is found that the average relaxation time and the peak amplitude of the RDF exhibit higher values for tumorous cells than normal cells and might be used as parameters to differentiate them and their associated tissues.

  3. Quantitative Microscopic Analysis of Plasma Membrane Receptor Dynamics in Living Plant Cells.

    Science.gov (United States)

    Luo, Yu; Russinova, Eugenia

    2017-01-01

    Plasma membrane-localized receptors are essential for cellular communication and signal transduction. In Arabidopsis thaliana, BRASSINOSTEROID INSENSITIVE1 (BRI1) is one of the receptors that is activated by binding to its ligand, the brassinosteroid (BR) hormone, at the cell surface to regulate diverse plant developmental processes. The availability of BRI1 in the plasma membrane is related to its signaling output and is known to be controlled by the dynamic endomembrane trafficking. Advances in fluorescence labeling and confocal microscopy techniques enabled us to gain a better understanding of plasma membrane receptor dynamics in living cells. Here we describe different quantitative microscopy methods to monitor the relative steady-state levels of the BRI1 protein in the plasma membrane of root epidermal cells and its relative exocytosis and recycling rates. The methods can be applied also to analyze similar dynamics of other plasma membrane-localized receptors.

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

    Science.gov (United States)

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

    2010-08-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-01-01

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

  6. Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy.

    Directory of Open Access Journals (Sweden)

    Sangeeta Nath

    Full Text Available INTRODUCTION: Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved. METHOD: Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties. RESULTS: The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii retention and spatial localization of chemical compounds vary within and between each cell line; and (iii the structural similarities of compounds can infer their non-specific binding properties. CONCLUSION: We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.

  7. A novel approach for the detection and genetic analysis of live melanoma circulating tumor cells.

    Directory of Open Access Journals (Sweden)

    Melody J Xu

    Full Text Available Circulating tumor cell (CTC detection and genetic analysis may complement currently available disease assessments in patients with melanoma to improve risk stratification and monitoring. We therefore sought to establish the feasibility of a telomerase-based assay for detecting and isolating live melanoma CTCs.The telomerase-based CTC assay utilizes an adenoviral vector that, in the presence of elevated human telomerase activity, drives the amplification of green fluorescent protein. Tumor cells are then identified via an image processing system. The protocol was tested on melanoma cells in culture or spiked into control blood, and on samples from patients with metastatic melanoma. Genetic analysis of the isolated melanoma CTCs was then performed for BRAF mutation status.The adenoviral vector was effective for all melanoma cell lines tested with sensitivity of 88.7% (95%CI 85.6-90.4% and specificity of 99.9% (95%CI 99.8-99.9%. In a pilot trial of patients with metastatic disease, CTCs were identified in 9 of 10 patients, with a mean of 6.0 CTCs/mL. At a cutoff of 1.1 CTCs/mL, the telomerase-based assay exhibits test performance of 90.0% sensitivity and 91.7% specificity. BRAF mutation analysis of melanoma cells isolated from culture or spiked control blood, or from pilot patient samples was found to match the known BRAF mutation status of the cell lines and primary tumors.To our knowledge, this is the first report of a telomerase-based assay effective for detecting and isolating live melanoma CTCs. These promising findings support further studies, including towards integrating into the management of patients with melanoma receiving multimodality therapy.

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

    Science.gov (United States)

    Barbarella, Giovanna; Di Maria, Francesca

    2015-08-18

    During the last few decades, multifunctional nano- and microfibers made of semiconducting π-conjugated oligomers and polymers have generated much interest because of a broad range of applications extending from sensing to bioelectronic devices and (opto)electronics. The simplest technique for the fabrication of these anisotropic supramolecular structures is to let the molecules do the work by spontaneous organization driven by the information encoded in their molecular structure. Oligothiophenes-semiconducting and fluorescent compounds that have been extensively investigated for applications in thin-film field-effect transistors and solar cells and to a lesser extent as dyes for fluorescent labeling of proteins, DNA, and live cells-are particularly suited as building blocks for supramolecular architectures because of the peculiar properties of the thiophene ring. Because of the great polarizability of sulfur outer-shell electrons and the consequent facile geometric deformability and adaptability of the ring to the environment, thiophene can generate multiple nonbonding interactions to promote non-covalent connections between blocks. Furthermore, sulfur can be hypervalent, i.e., it can accommodate more than the eight electrons normally associated with s and p shells. Hypervalent oligothiophene-S,S-dioxides whose oxygen atoms can be involved in hydrogen bonding have been synthesized. These compounds are amphiphilic, and some of them are able to spontaneously cross the membrane of live cells. Hypervalent nonbonding interactions of divalent sulfur, defined as weak coordination to a proximate nitrogen or oxygen, have also been invoked in the solid-state packing of many organic molecules and in the architecture of proteins. In this Account, we describe two different types of thiophene-based building blocks that can induce the spontaneous formation of nanostructured microfibers in very different environments. The first, based on the synthesis of "sulfur

  9. Molecular Theory of the Living Cell Concepts, Molecular Mechanisms, and Biomedical Applications

    CERN Document Server

    Ji, Sungchul

    2012-01-01

    This book presents a comprehensive molecular theory of the living cell based on over thirty concepts, principles and laws imported from thermodynamics, statistical mechanics, quantum mechanics, chemical kinetics, informatics, computer science, linguistics, semiotics, and philosophy. The author formulates physically, chemically and enzymologically realistic molecular mechanisms to account for the basic living processes such as ligand-receptor interactions, protein folding, single-molecule enzymic catalysis, force-generating mechanisms in molecular motors, signal transduction, regulation of the genome-wide RNA metabolism, morphogenesis, the micro-macro coupling in coordination dynamics, the origin of life, and the mechanisms of biological evolution itself. Possible solutions to basic and practical problems facing contemporary biology and biomedical sciences have been suggested, including pharmacotheragnostics and personalized medicine.

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

    Science.gov (United States)

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

    2017-09-15

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

  11. Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

    Science.gov (United States)

    Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

    2018-01-01

    Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

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

    Science.gov (United States)

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

    2010-05-01

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

  13. The effects of UV irradiation and gas plasma treatment on living mammalian cells and bacteria: a comparative approach

    NARCIS (Netherlands)

    Sosnin, E.A.; Stoffels - Adamowicz, E.; Erofeev, M.V.; Kieft, I.E.; Kunts, S.E.

    2004-01-01

    Living mammalian cells and bacteria were exposed to irradiation from narrow-band UV lamps and treated with a nonthermal gas plasma (plasma needle). The model systems were: Chinese Hamster Ovary (CHO-K1) cells (fibroblasts) and Escherichia Coli bacteria. UV irradiation can lead to cell death

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

    Science.gov (United States)

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

    2013-11-18

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

  15. A small molecular pH-dependent fluorescent probe for cancer cell imaging in living cell.

    Science.gov (United States)

    Ma, Junbao; Li, Wenqi; Li, Juanjuan; Shi, Rongguang; Yin, Gui; Wang, Ruiyong

    2018-05-15

    A novel pH-dependent two-photon fluorescent molecular probe ABMP has been prepared based on the fluorophore of 2, 4, 6-trisubstituted pyridine. The probe has an absorption wavelength at 354 nm and corresponding emission wavelength at 475 nm with the working pH range from 2.20 to 7.00, especially owning a good liner response from pH = 2.40 to pH = 4.00. ABMP also has excellent reversibility, photostability and selectivity which promotes its ability in analytical application. The probe can be excited with a two-photon fluorescence microscopy and the fluorescence cell imaging indicated that the probe can distinguish Hela cancer cells out of normal cells with a two-photon fluorescence microscopy which suggested its potential application in tumor cell detection. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Live-cell FRET imaging reveals clustering of the prion protein at the cell surface induced by infectious prions.

    Science.gov (United States)

    Tavares, Evandro; Macedo, Joana A; Paulo, Pedro M R; Tavares, Catarina; Lopes, Carlos; Melo, Eduardo P

    2014-07-01

    Prion diseases are associated to the conversion of the prion protein into a misfolded pathological isoform. The mechanism of propagation of protein misfolding by protein templating remains largely unknown. Neuroblastoma cells were transfected with constructs of the prion protein fused to both CFP-GPI-anchored and to YFP-GPI-anchored and directed to its cell membrane location. Live-cell FRET imaging between the prion protein fused to CFP or YFP was measured giving consistent values of 10±2%. This result was confirmed by fluorescence lifetime imaging microscopy and indicates intermolecular interactions between neighbor prion proteins. In particular, considering that a maximum FRET efficiency of 17±2% was determined from a positive control consisting of a fusion CFP-YFP-GPI-anchored. A stable cell clone expressing the two fusions containing the prion protein was also selected to minimize cell-to-cell variability. In both, stable and transiently transfected cells, the FRET efficiency consistently increased in the presence of infectious prions - from 4±1% to 7±1% in the stable clone and from 10±2% to 16±1% in transiently transfected cells. These results clearly reflect an increased clustering of the prion protein on the membrane in the presence of infectious prions, which was not observed in negative control using constructs without the prion protein and upon addition of non-infected brain. Our data corroborates the recent view that the primary site for prion conversion is the cell membrane. Since our fluorescent cell clone is not susceptible to propagate infectivity, we hypothesize that the initial event of prion infectivity might be the clustering of the GPI-anchored prion protein. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Live-cell fluorescent microscopy platforms for real-time monitoring of polyplex-cell interaction

    DEFF Research Database (Denmark)

    Parhamifar, Ladan; Wu, LinPing; Andersen, Helene

    2014-01-01

    A myriad of cationic polymeric delivery vehicles are currently being developed with the aim of transporting various forms of nucleic acids to mammalian cells. The complexes between polycations and nucleic acids are referred to as polyplexes. The screening for successful polyplex candidates requir...... of performance and intracellular trafficking of polyplexes as well as for assessing cell functionality. This review highlights the application of some of the most promising fluorescent microscopy platforms in relation to polyplex-mediated transfection processes....

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

    Directory of Open Access Journals (Sweden)

    Tomomi Ando

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

  19. Intermittency in 197Au fragmentation

    International Nuclear Information System (INIS)

    Dabrowska, A.; Holynski, R.; Olszewski, A.; Szarska, M.; Wilczynska, B.; Wolter, W.; Wosiek, B.; Cherry, M.L.; Deines-Jones, P.; Jones, W.V.; Sengupta, K.; Wefel, B.

    1995-07-01

    The concept of factorial moments was applied to an analysis of the dynamical fluctuations in the charge distributions of the fragments emitted from gold nuclei with energies 10.6 and < 1.0 GeV/n interacting with emulsion nuclei. Clear evidence for intermittent fluctuations has been found in an analysis using all the particles released from the gold projectile, with a stronger effect observed below 1 GeV/n than at 10.6 GeV/n. For the full data sets, however, the intermittency effect was found to be very sensitive to the singly charged particles, and neglecting these particles strongly reduces the intermittency signal. When the analysis is restricted to the multiply charged fragments, an intermittency effect is revealed only for multifragmentation events, although one that is enhanced as compared to the analysis of all, singly and multiply charged, particles. The properties of the anomalous fractal dimensions suggest a sequential decay mechanism, rather than the existence of possible critical behaviour in the process of nuclear fragmentation. The likely influence of the charge conservation effects and the finite size of decaying systems on the observed intermittency signals was pointed out. (author). 37 refs, 9 figs, 5 tabs

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yusuke Niino

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

  2. Real-time visualization of macromolecule uptake by epidermal Langerhans cells in living animals.

    Science.gov (United States)

    Frugé, Rachel E; Krout, Colleen; Lu, Ran; Matsushima, Hironori; Takashima, Akira

    2012-03-01

    As a skin-resident member of the dendritic cell family, Langerhans cells (LCs) are generally regarded to function as professional antigen-presenting cells. Here we report a simple method to visualize the endocytotic activity of LCs in living animals. BALB/c mice received subcutaneous injection of FITC-conjugated dextran (DX) probes into the ear skin and were then examined under confocal microscopy. Large numbers of FITC(+) epidermal cells became detectable 12-24 hours after injection as background fluorescence signals began to disappear. Most (>90%) of the FITC(+) epidermal cells expressed Langerin, and >95% of Langerin(+) epidermal cells exhibited significant FITC signals. To assess intracellular localization, Alexa Fluor 546-conjugated DX