Video-rate confocal microscopy for single-molecule imaging in live cells and superresolution fluorescence imaging.

Science.gov (United States)

Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

2012-10-17

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0-85 μm from the surface of a coverglass. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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.

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.

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...

Manipulation and Motion of Organelles and Single Molecules in Living Cells

DEFF Research Database (Denmark)

Norregaard, Kamilla; Metzler, Ralf; Ritter, Christine M.

2017-01-01

used force spectroscopy techniques, namely optical tweezers, magnetic tweezers, and atomic force microscopy, are described in detail, and their strength and limitations related to in vivo experiments are discussed. Finally, recent exciting discoveries within the field of in vivo manipulation...... driving many cellular processes. The forces on a molecular scale are exactly in the range that can be manipulated and probed with single molecule force spectroscopy. The natural environment of a biomolecule is inside a living cell, hence, this is the most relevant environment for probing their function...

Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy

Directory of Open Access Journals (Sweden)

Bjoern Traenkle

2017-08-01

Full Text Available Single-domain antibodies (sdAbs have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy.

Science.gov (United States)

Traenkle, Bjoern; Rothbauer, Ulrich

2017-01-01

Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies) have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

Exploring Transduction Mechanisms of Protein Transduction Domains (PTDs in Living Cells Utilizing Single-Quantum Dot Tracking (SQT Technology

Directory of Open Access Journals (Sweden)

Yasuhiro Suzuki

2012-01-01

Full Text Available Specific protein domains known as protein transduction domains (PTDs can permeate cell membranes and deliver proteins or bioactive materials into living cells. Various approaches have been applied for improving their transduction efficacy. It is, therefore, crucial to clarify the entry mechanisms and to identify the rate-limiting steps. Because of technical limitations for imaging PTD behavior on cells with conventional fluorescent-dyes, how PTDs enter the cells has been a topic of much debate. Utilizing quantum dots (QDs, we recently tracked the behavior of PTD that was derived from HIV-1 Tat (TatP in living cells at the single-molecule level with 7-nm special precision. In this review article, we initially summarize the controversy on TatP entry mechanisms; thereafter, we will focus on our recent findings on single-TatP-QD tracking (SQT, to identify the major sequential steps of intracellular delivery in living cells and to discuss how SQT can easily provide direct information on TatP entry mechanisms. As a primer for SQT study, we also discuss the latest findings on single particle tracking of various molecules on the plasma membrane. Finally, we discuss the problems of QDs and the challenges for the future in utilizing currently available QD probes for SQT. In conclusion, direct identification of the rate-limiting steps of PTD entry with SQT should dramatically improve the methods for enhancing transduction efficiency.

Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells

Energy Technology Data Exchange (ETDEWEB)

McFadden, Conor H. [Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Hallacy, Timothy M. [Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Department of Physics and Astronomy, Rice University, Houston, Texas (United States); Flint, David B. [Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas (United States); Granville, Dal A. [Department of Medical Physics, The Ottawa Hospital Cancer Centre, Ottawa, Ontario (Canada); Asaithamby, Aroumougame [Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Centre, Dallas, Texas (United States); Sahoo, Narayan [Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Akselrod, Mark S. [Crystal Growth Division, Landauer, Inc, Stillwater, Oklahoma (United States); Sawakuchi, Gabriel O., E-mail: gsawakuchi@mdanderson.org [Department of Radiation Physics, The University of Texas MD Anderson Cancer Center, Houston, Texas (United States); Graduate School of Biomedical Sciences, The University of Texas, Houston, Texas (United States)

2016-09-01

Purpose: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury. Methods and Materials: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of ∼1 × 10{sup 6} protons/cm{sup 2}. Results: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation. Conclusions: This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams.

Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy

OpenAIRE

Traenkle, Bjoern; Rothbauer, Ulrich

2017-01-01

Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies)...

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.

Probing bacterial adhesion at the single-cell level

DEFF Research Database (Denmark)

Zeng, Guanghong; Müller, Torsten; Meyer, Rikke Louise

be considered. We have developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion by force spectroscopy using atomic force microscopy (AFM). A single-cell probe was readily made by picking up a bacterial cell from a glass surface by approaching a tipless AFM...... cantilever coated with the commercial cell adhesive CellTakTM. We applied the method to study adhesion of living cells to abiotic surfaces at the single-cell level. Immobilisation of single bacterial cells to the cantilever was stable for several hours, and viability was confirmed by Live/Dead staining...... on the adhesion force, we explored the bond formation and adhesive strength of four different bacterial strains towards three abiotic substrates with variable hydrophobicity and surface roughness. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and time...

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.

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.

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.

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

International Nuclear Information System (INIS)

Brameshuber, M.

2009-01-01

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

Cell biochemistry studied by single-molecule imaging.

Science.gov (United States)

Mashanov, G I; Nenasheva, T A; Peckham, M; Molloy, J E

2006-11-01

Over the last decade, there have been remarkable developments in live-cell imaging. We can now readily observe individual protein molecules within living cells and this should contribute to a systems level understanding of biological pathways. Direct observation of single fluorophores enables several types of molecular information to be gathered. Temporal and spatial trajectories enable diffusion constants and binding kinetics to be deduced, while analyses of fluorescence lifetime, intensity, polarization or spectra give chemical and conformational information about molecules in their cellular context. By recording the spatial trajectories of pairs of interacting molecules, formation of larger molecular complexes can be studied. In the future, multicolour and multiparameter imaging of single molecules in live cells will be a powerful analytical tool for systems biology. Here, we discuss measurements of single-molecule mobility and residency at the plasma membrane of live cells. Analysis of diffusional paths at the plasma membrane gives information about its physical properties and measurement of temporal trajectories enables rates of binding and dissociation to be derived. Meanwhile, close scrutiny of individual fluorophore trajectories enables ideas about molecular dimerization and oligomerization related to function to be tested directly.

Measuring single-cell density.

Science.gov (United States)

Grover, William H; Bryan, Andrea K; Diez-Silva, Monica; Suresh, Subra; Higgins, John M; Manalis, Scott R

2011-07-05

We have used a microfluidic mass sensor to measure the density of single living cells. By weighing each cell in two fluids of different densities, our technique measures the single-cell mass, volume, and density of approximately 500 cells per hour with a density precision of 0.001 g mL(-1). We observe that the intrinsic cell-to-cell variation in density is nearly 100-fold smaller than the mass or volume variation. As a result, we can measure changes in cell density indicative of cellular processes that would be otherwise undetectable by mass or volume measurements. Here, we demonstrate this with four examples: identifying Plasmodium falciparum malaria-infected erythrocytes in a culture, distinguishing transfused blood cells from a patient's own blood, identifying irreversibly sickled cells in a sickle cell patient, and identifying leukemia cells in the early stages of responding to a drug treatment. These demonstrations suggest that the ability to measure single-cell density will provide valuable insights into cell state for a wide range of biological processes.

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.

QUANTITATION OF INTRACELLULAR NAD(P)H IN LIVING CELLS CAN MONITOR AN IMBALANCE OF DNA SINGLE STRAND BREAK REPAIR IN REAL TIME

Science.gov (United States)

Quantitation of intracellular NAD(P)H in living cells can monitor an imbalance of DNA single strand break repair in real time.ABSTRACTDNA single strand breaks (SSBs) are one of the most frequent DNA lesions in genomic DNA generated either by oxidative stress or du...

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.

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.

Convergence of lateral dynamic measurements in the plasma membrane of live cells from single particle tracking and STED-FCS

DEFF Research Database (Denmark)

Lagerholm, B. Christoffer; Andrade, Débora M.; Clausen, Mathias P.

2017-01-01

Fluorescence correlation spectroscopy (FCS) in combination with the super-resolution imaging method STED (STED-FCS), and single-particle tracking (SPT) are able to directly probe the lateral dynamics of lipids and proteins in the plasma membrane of live cells at spatial scales much below the diff...

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.

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.

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

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

Single-cell manipulation and DNA delivery technology using atomic force microscopy and nanoneedle.

Science.gov (United States)

Han, Sung-Woong; Nakamura, Chikashi; Miyake, Jun; Chang, Sang-Mok; Adachi, Taiji

2014-01-01

The recent single-cell manipulation technology using atomic force microscopy (AFM) not only allows high-resolution visualization and probing of biomolecules and cells but also provides spatial and temporal access to the interior of living cells via the nanoneedle technology. Here we review the development and application of single-cell manipulations and the DNA delivery technology using a nanoneedle. We briefly describe various DNA delivery methods and discuss their advantages and disadvantages. Fabrication of the nanoneedle, visualization of nanoneedle insertion into living cells, DNA modification on the nanoneedle surface, and the invasiveness of nanoneedle insertion into living cells are described. Different methods of DNA delivery into a living cell, such as lipofection, microinjection, and nanoneedles, are then compared. Finally, single-cell diagnostics using the nanoneedle and the perspectives of the nanoneedle technology are outlined. The nanoneedle-based DNA delivery technology provides new opportunities for efficient and specific introduction of DNA and other biomolecules into precious living cells with a high spatial resolution within a desired time frame. This technology has the potential to be applied for many basic cellular studies and for clinical studies such as single-cell diagnostics.

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

DEFF Research Database (Denmark)

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

be considered. We have therefore developed a simple and versatile method to make single-cell bacterial probes for measuring single cell adhesion with atomic force microscopy (AFM).[1] A single-cell probe was readily made by picking up a bacterial cell from a glass surface using a tipless AFM cantilever coated...... random immobilization is obtained by submerging the cantilever in a bacterial suspension. The reported method provides a general platform for investigating single cell interactions of bacteria with different surfaces and other cells by AFM force spectroscopy, thus improving our understanding....... The strain-dependent susceptibility to bacterial colonization on conventional PLL-g-PEG illustrates how bacterial diversity challenges development of “universal” antifouling coatings, and AFM single-cell force spectroscopy was proven to be a powerful tool to provide insights into the molecular mechanisms...

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

Real-Time Intracellular Measurements of ROS and RNS in Living Cells with Single Core-Shell Nanowire Electrodes.

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Zhang, Xin-Wei; Qiu, Quan-Fa; Jiang, Hong; Zhang, Fu-Li; Liu, Yan-Lin; Amatore, Christian; Huang, Wei-Hua

2017-10-09

Nanoelectrodes allow precise and quantitative measurements of important biological processes at the single living-cell level in real time. Cylindrical nanowire electrodes (NWEs) required for intracellular measurements create a great challenge for achieving excellent electrochemical and mechanical performances. Herein, we present a facile and robust solution to this problem based on a unique SiC-core-shell design to produce cylindrical NWEs with superior mechanical toughness provided by the SiC nano-core and an excellent electrochemical performance provided by the ultrathin carbon shell that can be used as such or platinized. The use of such NWEs for biological applications is illustrated by the first quantitative measurements of ROS/RNS in individual phagolysosomes of living macrophages. As the shell material can be varied to meet any specific detection purpose, this work opens up new opportunities to monitor quantitatively biological functions occurring inside cells and their organelles. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Single ovalbumin molecules exploring nucleoplasm and nucleoli of living cell nuclei.

Science.gov (United States)

Speil, Jasmin; Kubitscheck, Ulrich

2010-03-01

The nucleus is the center of direction and coordination of the cell's metabolic and reproductive activities and contains numerous functionally specialized domains. These subnuclear structures are not delimited by membranes like cytoplasmic organelles and their function is only poorly understood. Here, we studied the most prominent nuclear domains, nucleoli and the remaining nucleoplasm. We used fluorescently labeled ovalbumin-ATTO647N, an inert protein, to examine their physical properties. This inert tracer was microinjected into the cytoplasm of HeLa cells, and after diffusion into the nucleus the tracer distribution and mobility in the two nuclear compartments was examined. Like many macromolecular probes ovalbumin was significantly less abundant in nucleoli compared to the nucleoplasm. High-speed fluorescence microscopy allowed visualizing and analyzing single tracer molecule trajectories within nucleoli and nucleoplasm. In accordance with previous studies we found that the viscosity of the nucleus is sevenfold higher than that of aqueous buffer. Notably, nucleoplasm and nucleoli did not significantly differ in viscosity, however, the fraction of slow or trapped molecules was higher in the nucleoplasm than in nucleoli (6% versus 0.2%). Surprisingly, even a completely inert molecule like ovalbumin showed at times short-lived binding events with a decay time of 8 ms in the nucleoplasm and even shorter-6.3 ms-within the nucleoli. Copyright 2009 Elsevier B.V. All rights reserved.

Revealing dynamically-organized receptor ion channel clusters in live cells by a correlated electric recording and super-resolution single-molecule imaging approach.

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Yadav, Rajeev; Lu, H Peter

2018-03-28

The N-methyl-d-aspartate (NMDA) receptor ion-channel is activated by the binding of ligands, along with the application of action potential, important for synaptic transmission and memory functions. Despite substantial knowledge of the structure and function, the gating mechanism of the NMDA receptor ion channel for electric on-off signals is still a topic of debate. We investigate the NMDA receptor partition distribution and the associated channel's open-close electric signal trajectories using a combined approach of correlating single-molecule fluorescence photo-bleaching, single-molecule super-resolution imaging, and single-channel electric patch-clamp recording. Identifying the compositions of NMDA receptors, their spatial organization and distributions over live cell membranes, we observe that NMDA receptors are organized inhomogeneously: nearly half of the receptor proteins are individually dispersed; whereas others exist in heterogeneous clusters of around 50 nm in size as well as co-localized within the diffraction limited imaging area. We demonstrate that inhomogeneous interactions and partitions of the NMDA receptors can be a cause of the heterogeneous gating mechanism of NMDA receptors in living cells. Furthermore, comparing the imaging results with the ion-channel electric current recording, we propose that the clustered NMDA receptors may be responsible for the variation in the current amplitude observed in the on-off two-state ion-channel electric signal trajectories. Our findings shed new light on the fundamental structure-function mechanism of NMDA receptors and present a conceptual advancement of the ion-channel mechanism in living cells.

Using Single-Protein Tracking to Study Cell Migration.

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Orré, Thomas; Mehidi, Amine; Massou, Sophie; Rossier, Olivier; Giannone, Grégory

2018-01-01

To get a complete understanding of cell migration, it is critical to study its orchestration at the molecular level. Since the recent developments in single-molecule imaging, it is now possible to study molecular phenomena at the single-molecule level inside living cells. In this chapter, we describe how such approaches have been and can be used to decipher molecular mechanisms involved in cell migration.

Mutation-Specific Mechanisms of Hyperactivation of Noonan Syndrome SOS Molecules Detected with Single-molecule Imaging in Living Cells.

Science.gov (United States)

Nakamura, Yuki; Umeki, Nobuhisa; Abe, Mitsuhiro; Sako, Yasushi

2017-10-26

Noonan syndrome (NS) is a congenital hereditary disorder associated with developmental and cardiac defects. Some patients with NS carry mutations in SOS, a guanine nucleotide exchange factor (GEF) for the small GTPase RAS. NS mutations have been identified not only in the GEF domain, but also in various domains of SOS, suggesting that multiple mechanisms disrupt SOS function. In this study, we examined three NS mutations in different domains of SOS to clarify the abnormality in its translocation to the plasma membrane, where SOS activates RAS. The association and dissociation kinetics between SOS tagged with a fluorescent protein and the living cell surface were observed in single molecules. All three mutants showed increased affinity for the plasma membrane, inducing excessive RAS signalling. However, the mechanisms by which their affinity was increased were specific to each mutant. Conformational disorder in the resting state, increased probability of a conformational change on the plasma membrane, and an increased association rate constant with the membrane receptor are the suggested mechanisms. These different properties cause the specific phenotypes of the mutants, which should be rescuable with different therapeutic strategies. Therefore, single-molecule kinetic analyses of living cells are useful for the pathological analysis of genetic diseases.

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.

Interrogation of inhibitor of nuclear factor κB α/nuclear factor κB (IκBα/NF-κB) negative feedback loop dynamics: from single cells to live animals in vivo.

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Moss, Britney L; Elhammali, Adnan; Fowlkes, Tiffanie; Gross, Shimon; Vinjamoori, Anant; Contag, Christopher H; Piwnica-Worms, David

2012-09-07

Full understanding of the biological significance of negative feedback processes requires interrogation at multiple scales as follows: in single cells, cell populations, and live animals in vivo. The transcriptionally coupled IκBα/NF-κB negative feedback loop, a pivotal regulatory node of innate immunity and inflammation, represents a model system for multiscalar reporters. Using a κB(5)→IκBα-FLuc bioluminescent reporter, we rigorously evaluated the dynamics of ΙκBα degradation and subsequent NF-κB transcriptional activity in response to diverse modes of TNFα stimulation. Modulating TNFα concentration or pulse duration yielded complex, reproducible, and differential ΙκBα dynamics in both cell populations and live single cells. Tremendous heterogeneity in the transcriptional amplitudes of individual responding cells was observed, which was greater than the heterogeneity in the transcriptional kinetics of responsive cells. Furthermore, administration of various TNFα doses in vivo generated ΙκBα dynamic profiles in the liver resembling those observed in single cells and populations of cells stimulated with TNFα pulses. This suggested that dose modulation of circulating TNFα was perceived by hepatocytes in vivo as pulses of increasing duration. Thus, a robust bioluminescent reporter strategy enabled rigorous quantitation of NF-κB/ΙκBα dynamics in both live single cells and cell populations and furthermore, revealed reproducible behaviors that informed interpretation of in vivo studies.

[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.

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.

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

iSBatch: a batch-processing platform for data analysis and exploration of live-cell single-molecule microscopy images and other hierarchical datasets.

Science.gov (United States)

Caldas, Victor E A; Punter, Christiaan M; Ghodke, Harshad; Robinson, Andrew; van Oijen, Antoine M

2015-10-01

Recent technical advances have made it possible to visualize single molecules inside live cells. Microscopes with single-molecule sensitivity enable the imaging of low-abundance proteins, allowing for a quantitative characterization of molecular properties. Such data sets contain information on a wide spectrum of important molecular properties, with different aspects highlighted in different imaging strategies. The time-lapsed acquisition of images provides information on protein dynamics over long time scales, giving insight into expression dynamics and localization properties. Rapid burst imaging reveals properties of individual molecules in real-time, informing on their diffusion characteristics, binding dynamics and stoichiometries within complexes. This richness of information, however, adds significant complexity to analysis protocols. In general, large datasets of images must be collected and processed in order to produce statistically robust results and identify rare events. More importantly, as live-cell single-molecule measurements remain on the cutting edge of imaging, few protocols for analysis have been established and thus analysis strategies often need to be explored for each individual scenario. Existing analysis packages are geared towards either single-cell imaging data or in vitro single-molecule data and typically operate with highly specific algorithms developed for particular situations. Our tool, iSBatch, instead allows users to exploit the inherent flexibility of the popular open-source package ImageJ, providing a hierarchical framework in which existing plugins or custom macros may be executed over entire datasets or portions thereof. This strategy affords users freedom to explore new analysis protocols within large imaging datasets, while maintaining hierarchical relationships between experiments, samples, fields of view, cells, and individual molecules.

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.).

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

Directory of Open Access Journals (Sweden)

Rosemary A Bamford

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

A micro-Raman study of live, single red blood cells (RBCs treated with AgNO3 nanoparticles.

Directory of Open Access Journals (Sweden)

Aseefhali Bankapur

Full Text Available Silver nanoparticles (Ag NPs are known to exhibit broad antimicrobial activity. However, such activity continues to raise concerns in the context of the interaction of such NPs with biomolecules. In a physiological environment NPs interact with individual biological cells either by penetrating through the cell membrane or by adhering to the membrane. We have explored the interaction of Ag NPs with single optically-trapped, live erythrocytes (red blood cells, RBCs using Raman Tweezers spectroscopy. Our experiments reveal that Ag NPs induce modifications within an RBC that appear to be irreversible. In particular we are able to identify that the heme conformation in an RBC transforms from the usual R-state (oxy-state to the T-state (deoxy-state. We rationalize our observations by proposing a model for the nanoparticle cytotoxicity pathway when the NP size is larger than the membrane pore size. We propose that the interaction of Ag NPs with the cell surface induces damage brought about by alteration of intracellular pH caused by the blockage of the cell membrane transport.

High resolution imaging of surface patterns of single bacterial cells

International Nuclear Information System (INIS)

Greif, Dominik; Wesner, Daniel; Regtmeier, Jan; Anselmetti, Dario

2010-01-01

We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.

Single Molecule Detection in Living Biological Cells using Carbon Nanotube Optical Probes

Science.gov (United States)

Strano, Michael

2009-03-01

Nanoscale sensing elements offer promise for single molecule analyte detection in physically or biologically constrained environments. Molecular adsorption can be amplified via modulation of sharp singularities in the electronic density of states that arise from 1D quantum confinement [1]. Single-walled carbon nanotubes (SWNT), as single molecule optical sensors [2-3], offer unique advantages such as photostable near-infrared (n-IR) emission for prolonged detection through biological media, single-molecule sensitivity and, nearly orthogonal optical modes for signal transduction that can be used to identify distinct classes of analytes. Selective binding to the SWNT surface is difficult to engineer [4]. In this lecture, we will briefly review the immerging field of fluorescent diagnostics using band gap emission from SWNT. In recent work, we demonstrate that even a single pair of SWNT provides at least four optical modes that can be modulated to uniquely fingerprint chemical agents by the degree to which they alter either the emission band intensity or wavelength. We validate this identification method in vitro by demonstrating detection and identification of six genotoxic analytes, including chemotherapeutic drugs and reactive oxygen species (ROS), which are spectroscopically differentiated into four distinct classes. We also demonstrate single-molecule sensitivity in detecting hydrogen peroxide, one of the most common genotoxins and an important cellular signal. Finally, we employ our sensing and fingerprinting method of these analytes in real time within live 3T3 cells, demonstrating the first multiplexed optical detection from a nanoscale biosensor and the first label-free tool to optically discriminate between genotoxins. We will also discuss our recent efforts to fabricate biomedical sensors for real time detection of glucose and other important physiologically relevant analytes in-vivo. The response of embedded SWNT in a swellable hydrogel construct to

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.

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.

Axial tomography in live cell laser microscopy

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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.

A precise pointing nanopipette for single-cell imaging via electroosmotic injection.

Science.gov (United States)

Lv, Jian; Qian, Ruo-Can; Hu, Yong-Xu; Liu, Shao-Chuang; Cao, Yue; Zheng, Yong-Jie; Long, Yi-Tao

2016-11-24

The precise transportation of fluorescent probes to the designated location in living cells is still a challenge. Here, we present a new addition to nanopipettes as a powerful tool to deliver fluorescent molecules to a given place in a single cell by electroosmotic flow, indicating favorable potential for further application in single-cell imaging.

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.

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

Real-time Molecular Study of Bystander Effects of Low dose Low LET radiation Using Living Cell Imaging and Nanoparticale Optics

Energy Technology Data Exchange (ETDEWEB)

Natarajan, Mohan [UT Health Science Center at San Antonio; Xu, Nancy R [Old Dominion University; Mohan, Sumathy [UT Health Science Center at San Antonio

2013-06-03

In this study two novel approaches are proposed to investigate precisely the low dose low LET radiation damage and its effect on bystander cells in real time. First, a flow shear model system, which would provide us a near in vivo situation where endothelial cells in the presence of extra cellular matrix experiencing continuous flow shear stress, will be used. Endothelial cells on matri-gel (simulated extra cellular matrix) will be subjected to physiological flow shear (that occurs in normal blood vessels). Second, a unique tool (Single nano particle/single live cell/single molecule microscopy and spectroscopy; Figure A) will be used to track the molecular trafficking by single live cell imaging. Single molecule chemical microscopy allows one to single out and study rare events that otherwise might be lost in assembled average measurement, and monitor many target single molecules simultaneously in real-time. Multi color single novel metal nanoparticle probes allow one to prepare multicolor probes (Figure B) to monitor many single components (events) simultaneously and perform multi-complex analysis in real-time. These nano-particles resist to photo bleaching and hence serve as probes for unlimited timeframe of analysis. Single live cell microscopy allows one to image many single cells simultaneously in real-time. With the combination of these unique tools, we will be able to study under near-physiological conditions the cellular and sub-cellular responses (even subtle changes at one molecule level) to low and very low doses of low LET radiation in real time (milli-second or nano-second) at sub-10 nanometer spatial resolution. This would allow us to precisely identify, at least in part, the molecular mediators that are responsible of radiation damage in the irradiated cells and the mediators that are responsible for initiating the signaling in the neighboring cells. Endothelial cells subjected to flow shear (2 dynes/cm2 or 16 dynes/cm2) and exposed to 0.1, 1 and 10

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.

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.

The nature and nurture of cell heterogeneity: accounting for macrophage gene-environment interactions with single-cell RNA-Seq.

Science.gov (United States)

Wills, Quin F; Mellado-Gomez, Esther; Nolan, Rory; Warner, Damien; Sharma, Eshita; Broxholme, John; Wright, Benjamin; Lockstone, Helen; James, William; Lynch, Mark; Gonzales, Michael; West, Jay; Leyrat, Anne; Padilla-Parra, Sergi; Filippi, Sarah; Holmes, Chris; Moore, Michael D; Bowden, Rory

2017-01-07

Single-cell RNA-Seq can be a valuable and unbiased tool to dissect cellular heterogeneity, despite the transcriptome's limitations in describing higher functional phenotypes and protein events. Perhaps the most important shortfall with transcriptomic 'snapshots' of cell populations is that they risk being descriptive, only cataloging heterogeneity at one point in time, and without microenvironmental context. Studying the genetic ('nature') and environmental ('nurture') modifiers of heterogeneity, and how cell population dynamics unfold over time in response to these modifiers is key when studying highly plastic cells such as macrophages. We introduce the programmable Polaris™ microfluidic lab-on-chip for single-cell sequencing, which performs live-cell imaging while controlling for the culture microenvironment of each cell. Using gene-edited macrophages we demonstrate how previously unappreciated knockout effects of SAMHD1, such as an altered oxidative stress response, have a large paracrine signaling component. Furthermore, we demonstrate single-cell pathway enrichments for cell cycle arrest and APOBEC3G degradation, both associated with the oxidative stress response and altered proteostasis. Interestingly, SAMHD1 and APOBEC3G are both HIV-1 inhibitors ('restriction factors'), with no known co-regulation. As single-cell methods continue to mature, so will the ability to move beyond simple 'snapshots' of cell populations towards studying the determinants of population dynamics. By combining single-cell culture, live-cell imaging, and single-cell sequencing, we have demonstrated the ability to study cell phenotypes and microenvironmental influences. It's these microenvironmental components - ignored by standard single-cell workflows - that likely determine how macrophages, for example, react to inflammation and form treatment resistant HIV reservoirs.

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.

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.

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

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.

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.

QSpec: online control and data analysis system for single-cell Raman spectroscopy

Directory of Open Access Journals (Sweden)

Lihui Ren

2014-06-01

Full Text Available Single-cell phenotyping is critical to the success of biological reductionism. Raman-activated cell sorting (RACS has shown promise in resolving the dynamics of living cells at the individual level and to uncover population heterogeneities in comparison to established approaches such as fluorescence-activated cell sorting (FACS. Given that the number of single-cells would be massive in any experiment, the power of Raman profiling technique for single-cell analysis would be fully utilized only when coupled with a high-throughput and intelligent process control and data analysis system. In this work, we established QSpec, an automatic system that supports high-throughput Raman-based single-cell phenotyping. Additionally, a single-cell Raman profile database has been established upon which data-mining could be applied to discover the heterogeneity among single-cells under different conditions. To test the effectiveness of this control and data analysis system, a sub-system was also developed to simulate the phenotypes of single-cells as well as the device features.

Using micro-patterned sensors and cell self-assembly for measuring the oxygen consumption rate of single cells

International Nuclear Information System (INIS)

Etzkorn, James R; Parviz, Babak A; Wu, Wen-Chung; Tian, Zhiyuan; Kim, Prince; Jang, Sei-Hum; Jen, Alex K-Y; Meldrum, Deirdre R

2010-01-01

We present a method for self-assembling arrays of live single cells on a glass chip using a photopatternable polymer to form micro-traps. We have studied the single-cell self-assembly method and optimized the process to obtain a 52% yield of single-trapped cells. We also report a method to measure the oxygen consumption rate of a single cell using micro-patterned sensors. These molecular oxygen sensors were fabricated around each micro-trap allowing optical interrogation of oxygen concentration in the immediate environment of the trapped cell. Micromachined micro-wells were then used to seal the trap, sensor and cell in order to determine the oxygen consumption rate of single cells. These techniques reported here add to the collection of tools for performing 'singe-cell' biology. An oxygen consumption rate of 1.05 ± 0.28 fmol min −1 was found for a data set consisting of 25 single A549 cells.

Detection and analysis of human serum albumin nanoparticles within phagocytic cells at the resolution of individual live cell or single 3D multicellular spheroid

Energy Technology Data Exchange (ETDEWEB)

Afrimzon, Elena; Zurgil, Naomi; Sobolev, Maria; Shafran, Yana [Bar-Ilan University, The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome (Israel); Langer, Klaus; Zlatev, Iavor [Westfälischen Wilhelms-Universität Münster, Institut für Pharmazeutische Technologie und Biopharmazie (Germany); Wronski, Robert; Windisch, Manfred [QPS Austria GmbH (Austria); Briesen, Hagen von [Fraunhofer Institute for Biomedical Engineering IBMT, Department of Cell Biology & Applied Virology (Germany); Schmidt, Reinhold [Medical University of Graz, Department of Neurology (Austria); Pietrzik, Claus [University Medical Center of the Johannes Gutenberg University of Mainz, Institute of Pathobiochemistry (Germany); Deutsch, Mordechai, E-mail: motti.jsc@gmail.com [Bar-Ilan University, The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome (Israel)

2015-12-15

Since nanoparticles (NPs) have shown great potential in various biomedical applications, live cell response to NPs should be thoroughly explored prior to their in vivo use. In the current study, live cell array (LCA) methodology and unique cell-based assays were used to study the interaction of magnetite (HSA-Mag NP) loaded human serum albumin NPs with phagocytic cells. The LCA enabled cell culturing during HSA-Mag NP accumulation and monolayer or spheroid formation, concomitantly with on-line monitoring of NP internalization. These platforms were also utilized for imaging intercellular links between living cells preloaded with HSA-Mag NP in 2D and 3D resolution. HSA-Mag NP uptake by cells was quantified by imaging, and analyzed using time-resolved measurements. Image analysis of the individual cells in cell populations showed accumulation of HSA-Mag NP by promonocytes and glial cells in a dose- and time-dependent manner. High variability of NP accumulation in individual cells within cell populations, as well as cell subgroups, was evident in both cell types. Following 24 h interaction, uptake of HSA-Mag NP was about 10 times more efficient in glial cells than in activated promonocytes. The presented assays may facilitate detection and analysis of the amount of NPs within individual cells, as well as the rate of NP accumulation and processing in different subsets of living cells. Such data are crucial for estimating predicted drug dosage delivered by NPs, as well as to study possible mechanisms for NP interference with live cells.

Transcription-factor-mediated DNA looping probed by high-resolution, single-molecule imaging in live E. coli cells.

Directory of Open Access Journals (Sweden)

Zach Hensel

Full Text Available DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The "genetic switch" of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences O(R and O(L (separated by 2.3 kb, mediated by the λ repressor CI (accession number P03034, play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms.

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

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.

Correlated receptor transport processes buffer single-cell heterogeneity.

Directory of Open Access Journals (Sweden)

Stefan M Kallenberger

2017-09-01

Full Text Available Cells typically vary in their response to extracellular ligands. Receptor transport processes modulate ligand-receptor induced signal transduction and impact the variability in cellular responses. Here, we quantitatively characterized cellular variability in erythropoietin receptor (EpoR trafficking at the single-cell level based on live-cell imaging and mathematical modeling. Using ensembles of single-cell mathematical models reduced parameter uncertainties and showed that rapid EpoR turnover, transport of internalized EpoR back to the plasma membrane, and degradation of Epo-EpoR complexes were essential for receptor trafficking. EpoR trafficking dynamics in adherent H838 lung cancer cells closely resembled the dynamics previously characterized by mathematical modeling in suspension cells, indicating that dynamic properties of the EpoR system are widely conserved. Receptor transport processes differed by one order of magnitude between individual cells. However, the concentration of activated Epo-EpoR complexes was less variable due to the correlated kinetics of opposing transport processes acting as a buffering system.

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)

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.

Long-term in vivo imaging of multiple organs at the single cell level.

Directory of Open Access Journals (Sweden)

Benny J Chen

Full Text Available Two-photon microscopy has enabled the study of individual cell behavior in live animals. Many organs and tissues cannot be studied, especially longitudinally, because they are located too deep, behind bony structures or too close to the lung and heart. Here we report a novel mouse model that allows long-term single cell imaging of many organs. A wide variety of live tissues were successfully engrafted in the pinna of the mouse ear. Many of these engrafted tissues maintained the normal tissue histology. Using the heart and thymus as models, we further demonstrated that the engrafted tissues functioned as would be expected. Combining two-photon microscopy with fluorescent tracers, we successfully visualized the engrafted tissues at the single cell level in live mice over several months. Four dimensional (three-dimensional (3D plus time information of individual cells was obtained from this imaging. This model makes long-term high resolution 4D imaging of multiple organs possible.

RoboSCell: An automated single cell arraying and analysis instrument

KAUST Repository

Sakaki, Kelly

2009-09-09

Single cell research has the potential to revolutionize experimental methods in biomedical sciences and contribute to clinical practices. Recent studies suggest analysis of single cells reveals novel features of intracellular processes, cell-to-cell interactions and cell structure. The methods of single cell analysis require mechanical resolution and accuracy that is not possible using conventional techniques. Robotic instruments and novel microdevices can achieve higher throughput and repeatability; however, the development of such instrumentation is a formidable task. A void exists in the state-of-the-art for automated analysis of single cells. With the increase in interest in single cell analyses in stem cell and cancer research the ability to facilitate higher throughput and repeatable procedures is necessary. In this paper, a high-throughput, single cell microarray-based robotic instrument, called the RoboSCell, is described. The proposed instrument employs a partially transparent single cell microarray (SCM) integrated with a robotic biomanipulator for in vitro analyses of live single cells trapped at the array sites. Cells, labeled with immunomagnetic particles, are captured at the array sites by channeling magnetic fields through encapsulated permalloy channels in the SCM. The RoboSCell is capable of systematically scanning the captured cells temporarily immobilized at the array sites and using optical methods to repeatedly measure extracellular and intracellular characteristics over time. The instrument\\'s capabilities are demonstrated by arraying human T lymphocytes and measuring the uptake dynamics of calcein acetoxymethylester-all in a fully automated fashion. © 2009 Springer Science+Business Media, LLC.

Single-Molecule Light-Sheet Imaging of Suspended T Cells.

Science.gov (United States)

Ponjavic, Aleks; McColl, James; Carr, Alexander R; Santos, Ana Mafalda; Kulenkampff, Klara; Lippert, Anna; Davis, Simon J; Klenerman, David; Lee, Steven F

2018-05-08

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose. Copyright © 2018. Published by Elsevier Inc.

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.

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.

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.

Living in single person households and the risk of isolation in later life

Directory of Open Access Journals (Sweden)

Laura Banks

2009-10-01

Full Text Available Data from the International Social Survey Programme (2001 was used to analyse the social networks of older people and whether living in single person households increased the risk of isolation. When comparing respondents with one or more adult children, there was no significant difference in the likelihood of experiencing familial isolation between people living in single person households and those living in larger households. A majority of those living in single person households had at least regular contact with a sibling, adult child or close friend and participated in a social organisation. Friends compensate to some extent for a lack of support from the family, although in southern and eastern European countries, other relatives appeared to be more important in support networks. People living in single person households were more likely to experience isolation, but this was largely related to advanced age and childlessness. Whilst a very small minority in Japan were living in single person households, they were significantly more likely to be severely isolated than those living in single person households in other countries.

Application of confocal Raman micro-spectroscopy for label-free monitoring of oxidative stress in living bronchial cells

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Surmacki, Jakub M.; Quirós Gonzalez, Isabel; Bohndiek, Sarah E.

2018-02-01

Oxidative stress in cancer is implicated in tumor progression, being associated with increased therapy resistance and metastasis. Conventional approaches for monitoring oxidative stress in tissue such as high-performance liquid chromatography and immunohistochemistry are bulk measurements and destroy the sample, meaning that longitudinal monitoring of cancer cell heterogeneity remains elusive. Raman spectroscopy has the potential to overcome this challenge, providing a chemically specific, label free readout from single living cells. Here, we applied a standardized protocol for label-free confocal Raman micro-spectroscopy in living cells to monitor oxidative stress in bronchial cells. We used a quartz substrate in a commercial cell chamber contained within a microscope incubator providing culture media for cell maintenance. We studied the effect of a potent reactive oxygen species inducer, tert-butyl hydroperoxide (TBHP), and antioxidant, N-acetyl-L-cysteine (NAC) on living cells from a human bronchial epithelial cells (HBEC). We found that the Raman bands corresponding to nucleic acids, proteins and lipids were significantly different (pmicro-spectroscopy may be able to monitor the biological impact of oxidative and reductive processes in cells, hence enabling longitudinal studies of oxidative stress in therapy resistance and metastasis at the single cell level.

Single-cell tracking reveals antibiotic-induced changes in mycobacterial energy metabolism.

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Maglica, Željka; Özdemir, Emre; McKinney, John D

2015-02-17

ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening. New antimicrobials are urgently needed to stem the rising tide of antibiotic-resistant bacteria. All antibiotics are expected to affect bacterial energy metabolism, directly or indirectly, yet tools to assess the impact of antibiotics on the ATP content of individual bacterial cells are lacking. The

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

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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.

Single-cell analysis of growth and cell division of the anaerobe Desulfovibrio vulgaris Hildenborough

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Anouchka eFievet

2015-12-01

Full Text Available Recent years have seen significant progress in understanding basic bacterial cell cycle properties such as cell growth and cell division. While characterization and regulation of bacterial cell cycle is quite well documented in the case of fast growing aerobic model organisms, no data has been so far reported for anaerobic bacteria. This lack of information in anaerobic microorganisms can mainly be explained by the absence of molecular and cellular tools such as single cell microscopy and fluorescent probes usable for anaerobes and essential to study cellular events and/or subcellular localization of the actors involved in cell cycle.In this study, single-cell microscopy has been adapted to study for the first time, in real time, the cell cycle of a bacterial anaerobe, Desulfovibrio vulgaris Hildenborough (DvH. This single-cell analysis provides mechanistic insights into the cell division cycle of DvH, which seems to be governed by the recently discussed so-called incremental model that generates remarkably homogeneous cell sizes. Furthermore, cell division was reversibly blocked during oxygen exposure. This may constitute a strategy for anaerobic cells to cope with transient exposure to oxygen that they may encounter in their natural environment, thereby contributing to their aerotolerance. This study lays the foundation for the first molecular, single-cell assay that will address factors that cannot otherwise be resolved in bulk assays and that will allow visualization of a wide range of molecular mechanisms within living anaerobic cells.

Single Cell Assay for Analyzing Single Cell Exosome and Endocrine Secretion and Cancer Markers

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Chiu, Yu-Jui

To understand the inhomogeneity of cells in biological systems, there is a growing demand for the capability to characterize the properties of individual single cells. Since single cell studies require continuous monitoring of the cell behaviors instead of a snapshot test at a single time point, an effective single-cell assay that can support time lapsed studies in a high throughput manner is desired. Most currently available single-cell technologies cannot provide proper environments to sustain cell growth and cannot provide, for appropriate cell types, proliferation of single cells and convenient, non-invasive tests of single cell behaviors from molecular markers. In this dissertation, I present a highly versatile single-cell assay that can accommodate different cellular types, enable easy and efficient single cell loading and culturing, and be suitable for the study of effects of in-vitro environmental factors in combination with drug screening. The salient features of the assay are the non-invasive collection and surveying of single cell secretions at different time points and massively parallel translocation of single cells by user defined criteria, producing very high compatibility to the downstream process such as single cell qPCR and sequencing. Above all, the acquired information is quantitative -- for example, one of the studies is measured by the number of exosomes each single cell secretes for a given time period. Therefore, our single-cell assay provides a convenient, low-cost, and enabling tool for quantitative, time lapsed studies of single cell properties.

Time-resolved, single-cell analysis of induced and programmed cell death via non-invasive propidium iodide and counterstain perfusion.

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Krämer, Christina E M; Wiechert, Wolfgang; Kohlheyer, Dietrich

2016-09-01

Conventional propidium iodide (PI) staining requires the execution of multiple steps prior to analysis, potentially affecting assay results as well as cell vitality. In this study, this multistep analysis method has been transformed into a single-step, non-toxic, real-time method via live-cell imaging during perfusion with 0.1 μM PI inside a microfluidic cultivation device. Dynamic PI staining was an effective live/dead analytical tool and demonstrated consistent results for single-cell death initiated by direct or indirect triggers. Application of this method for the first time revealed the apparent antibiotic tolerance of wild-type Corynebacterium glutamicum cells, as indicated by the conversion of violet fluorogenic calcein acetoxymethyl ester (CvAM). Additional implementation of this method provided insight into the induced cell lysis of Escherichia coli cells expressing a lytic toxin-antitoxin module, providing evidence for non-lytic cell death and cell resistance to toxin production. Finally, our dynamic PI staining method distinguished necrotic-like and apoptotic-like cell death phenotypes in Saccharomyces cerevisiae among predisposed descendants of nutrient-deprived ancestor cells using PO-PRO-1 or green fluorogenic calcein acetoxymethyl ester (CgAM) as counterstains. The combination of single-cell cultivation, fluorescent time-lapse imaging, and PI perfusion facilitates spatiotemporally resolved observations that deliver new insights into the dynamics of cellular behaviour.

Modulation of protein properties in living cells using nanobodies.

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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.

A Checklist for Successful Quantitative Live Cell Imaging in Systems Biology

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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

Single molecule microscopy in 3D cell cultures and tissues.

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Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

2014-12-15

From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

Fast automatic quantitative cell replication with fluorescent live cell imaging

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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.

Convergence of lateral dynamic measurements in the plasma membrane of live cells from single particle tracking and STED-FCS

International Nuclear Information System (INIS)

Lagerholm, B Christoffer; Eggeling, Christian; Andrade, Débora M; Clausen, Mathias P

2017-01-01

Fluorescence correlation spectroscopy (FCS) in combination with the super-resolution imaging method STED (STED-FCS), and single-particle tracking (SPT) are able to directly probe the lateral dynamics of lipids and proteins in the plasma membrane of live cells at spatial scales much below the diffraction limit of conventional microscopy. However, a major disparity in interpretation of data from SPT and STED-FCS remains, namely the proposed existence of a very fast (unhindered) lateral diffusion coefficient, ⩾5 µ m 2 s −1 , in the plasma membrane of live cells at very short length scales, ≈⩽ 100 nm, and time scales, ≈1–10 ms. This fast diffusion coefficient has been advocated in several high-speed SPT studies, for lipids and membrane proteins alike, but the equivalent has not been detected in STED-FCS measurements. Resolving this ambiguity is important because the assessment of membrane dynamics currently relies heavily on SPT for the determination of heterogeneous diffusion. A possible systematic error in this approach would thus have vast implications in this field. To address this, we have re-visited the analysis procedure for SPT data with an emphasis on the measurement errors and the effect that these errors have on the measurement outputs. We subsequently demonstrate that STED-FCS and SPT data, following careful consideration of the experimental errors of the SPT data, converge to a common interpretation which for the case of a diffusing phospholipid analogue in the plasma membrane of live mouse embryo fibroblasts results in an unhindered, intra-compartment, diffusion coefficient of  ≈0.7–1.0 µ m 2 s −1 , and a compartment size of about 100–150 nm. (topical review)

Live cell imaging at the Munich ion microbeam SNAKE - a status report.

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Drexler, Guido A; Siebenwirth, Christian; Drexler, Sophie E; Girst, Stefanie; Greubel, Christoph; Dollinger, Günther; Friedl, Anna A

2015-02-18

Ion microbeams are important tools in radiobiological research. Still, the worldwide number of ion microbeam facilities where biological experiments can be performed is limited. Even fewer facilities combine ion microirradiation with live-cell imaging to allow microscopic observation of cellular response reactions starting very fast after irradiation and continuing for many hours. At SNAKE, the ion microbeam facility at the Munich 14 MV tandem accelerator, a large variety of biological experiments are performed on a regular basis. Here, recent developments and ongoing research projects at the ion microbeam SNAKE are presented with specific emphasis on live-cell imaging experiments. An overview of the technical details of the setup is given, including examples of suitable biological samples. By ion beam focusing to submicrometer beam spot size and single ion detection it is possible to target subcellular structures with defined numbers of ions. Focusing of high numbers of ions to single spots allows studying the influence of high local damage density on recruitment of damage response proteins.

Innovative molecular-based fluorescent nanoparticles for multicolor single particle tracking in cells

International Nuclear Information System (INIS)

Daniel, Jonathan; Blanchard-Desce, Mireille; Godin, Antoine G; Palayret, Matthieu; Lounis, Brahim; Cognet, Laurent

2016-01-01

Based on an original molecular-based design, we present bright and photostable fluorescent organic nanoparticles (FONs) showing excellent colloidal stability in various aqueous environments. Complementary near-infrared emitting and green emitting FONs were prepared using a simple, fast and robust protocol. Both types of FONs could be simultaneously imaged at the single-particle level in solution as well as in biological environments using a monochromatic excitation and a dual-color fluorescence microscope. No evidence of acute cytotoxicity was found upon incubation of live cells with mixed solutions of FONs, and both types of nanoparticles were found internalized in the cells where their motion could be simultaneously tracked at video-rate up to minutes. These fluorescent organic nanoparticles open a novel non-toxic alternative to existing nanoparticles for imaging biological structures, compatible with live-cell experiments and specially fitted for multicolor single particle tracking. (paper)

Reproducible fashion of the HSP70B' promoter-induced cytotoxic response on a live cell-based biosensor by cell cycle synchronization.

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Migita, Satoshi; Wada, Ken-Ichi; Taniguchi, Akiyoshi

2010-10-15

Live cell-based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high-throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell-based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum-starved conditions and we then investigated the cadmium chloride-induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell-based biosensors with microdevices, such as a single cell array. Copyright 2010 Wiley Periodicals, Inc.

Single-cell nanotoxicity assays of superparamagnetic iron oxide nanoparticles.

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Eustaquio, Trisha; Leary, James F

2012-01-01

Properly evaluating the nanotoxicity of nanoparticles involves much more than bulk-cell assays of cell death by necrosis. Cells exposed to nanoparticles may undergo repairable oxidative stress and DNA damage or be induced into apoptosis. Exposure to nanoparticles may cause the cells to alter their proliferation or differentiation or their cell-cell signaling with neighboring cells in a tissue. Nanoparticles are usually more toxic to some cell subpopulations than others, and toxicity often varies with cell cycle. All of these facts dictate that any nanotoxicity assay must be at the single-cell level and must try whenever feasible and reasonable to include many of these other factors. Focusing on one type of quantitative measure of nanotoxicity, we describe flow and scanning image cytometry approaches to measuring nanotoxicity at the single-cell level by using a commonly used assay for distinguishing between necrotic and apoptotic causes of cell death by one type of nanoparticle. Flow cytometry is fast and quantitative, provided that the cells can be prepared into a single-cell suspension for analysis. But when cells cannot be put into suspension without altering nanotoxicity results, or if morphology, attachment, and stain location are important, a scanning image cytometry approach must be used. Both methods are described with application to a particular type of nanoparticle, a superparamagnetic iron oxide nanoparticle (SPION), as an example of how these assays may be applied to the more general problem of determining the effects of nanomaterial exposure to living cells.

Live cell imaging compatible immobilization of Chlamydomonas reinhardtii in microfluidic platform for biodiesel research.

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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.

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

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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.

Superresolution imaging in live Caulobacter crescentus cells using photoswitchable enhanced yellow fluorescent protein

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Biteen, Julie S.; Thompson, Michael A.; Tselentis, Nicole K.; Shapiro, Lucy; Moerner, W. E.

2009-02-01

Recently, photoactivation and photoswitching were used to control single-molecule fluorescent labels and produce images of cellular structures beyond the optical diffraction limit (e.g., PALM, FPALM, and STORM). While previous live-cell studies relied on sophisticated photoactivatable fluorescent proteins, we show in the present work that superresolution imaging can be performed with fusions to the commonly used fluorescent protein EYFP. Rather than being photoactivated, however, EYFP can be reactivated with violet light after apparent photobleaching. In each cycle after initial imaging, only a sparse subset fluorophores is reactivated and localized, and the final image is then generated from the measured single-molecule positions. Because these methods are based on the imaging nanometer-sized single-molecule emitters and on the use of an active control mechanism to produce sparse sub-ensembles, we suggest the phrase "Single-Molecule Active-Control Microscopy" (SMACM) as an inclusive term for this general imaging strategy. In this paper, we address limitations arising from physiologically imposed upper boundaries on the fluorophore concentration by employing dark time-lapse periods to allow single-molecule motions to fill in filamentous structures, increasing the effective labeling concentration while localizing each emitter at most once per resolution-limited spot. We image cell-cycle-dependent superstructures of the bacterial actin protein MreB in live Caulobacter crescentus cells with sub-40-nm resolution for the first time. Furthermore, we quantify the reactivation quantum yield of EYFP, and find this to be 1.6 x 10-6, on par with conventional photoswitchable fluorescent proteins like Dronpa. These studies show that EYFP is a useful emitter for in vivo superresolution imaging of intracellular structures in bacterial cells.

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)

Single-cell printing to form three-dimensional lines of olfactory ensheathing cells

International Nuclear Information System (INIS)

Othon, Christina M; Ringeisen, Bradley R; Wu Xingjia; Anders, Juanita J

2008-01-01

Biological laser printing (BioLP(TM)) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes (∼μLs) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 μm, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth

Surface-modified CMOS IC electrochemical sensor array targeting single chromaffin cells for highly parallel amperometry measurements.

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Huang, Meng; Delacruz, Joannalyn B; Ruelas, John C; Rathore, Shailendra S; Lindau, Manfred

2018-01-01

Amperometry is a powerful method to record quantal release events from chromaffin cells and is widely used to assess how specific drugs modify quantal size, kinetics of release, and early fusion pore properties. Surface-modified CMOS-based electrochemical sensor arrays allow simultaneous recordings from multiple cells. A reliable, low-cost technique is presented here for efficient targeting of single cells specifically to the electrode sites. An SU-8 microwell structure is patterned on the chip surface to provide insulation for the circuitry as well as cell trapping at the electrode sites. A shifted electrode design is also incorporated to increase the flexibility of the dimension and shape of the microwells. The sensitivity of the electrodes is validated by a dopamine injection experiment. Microwells with dimensions slightly larger than the cells to be trapped ensure excellent single-cell targeting efficiency, increasing the reliability and efficiency for on-chip single-cell amperometry measurements. The surface-modified device was validated with parallel recordings of live chromaffin cells trapped in the microwells. Rapid amperometric spikes with no diffusional broadening were observed, indicating that the trapped and recorded cells were in very close contact with the electrodes. The live cell recording confirms in a single experiment that spike parameters vary significantly from cell to cell but the large number of cells recorded simultaneously provides the statistical significance.

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

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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.

Single Motherhood, Living Arrangements, and Time With Children in Japan.

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Raymo, James M; Park, Hyunjoon; Iwasawa, Miho; Zhou, Yanfei

2014-08-01

The authors examined relationships between single parenthood and mothers' time with children in Japan. Using data from the 2011 National Survey of Households with Children (N = 1,926), they first demonstrate that time spent with children and the frequency of shared dinners are significantly lower for single mothers than for their married counterparts. For single mothers living alone, less time with children reflects long work hours and work-related stress. Single mothers coresiding with parents spend less time with children and eat dinner together less frequently than either married mothers or their unmarried counterparts not living with parents, net of (grand)parental support, work hours, income, and stress. The findings suggest that rising divorce rates and associated growth in single-mother families may have a detrimental impact on parents' time with children in Japan and that the relatively high prevalence of intergenerational coresidence among single mothers may do little to temper this impact.

Single-cell vs. bulk activity properties of coastal bacterioplankton over an annual cycle in a temperate ecosystem.

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Morán, Xosé Anxelu G; Calvo-Díaz, Alejandra

2009-01-01

The connections between single-cell activity properties of heterotrophic planktonic bacteria and whole community metabolism are still poorly understood. Here, we show flow cytometry single-cell analysis of membrane-intact (live), high nucleic acid (HNA) content and actively respiring (CTC+) bacteria with samples collected monthly during 2006 in northern Spain coastal waters. Bulk activity was assessed by measuring 3H-Leucine incorporation and specific growth rates. Consistently, different single-cell relative abundances were found, with 60-100% for live, 30-84% for HNA and 0.2-12% for CTC+ cells. Leucine incorporation rates (2-153 pmol L(-1) h(-1)), specific growth rates (0.01-0.29 day(-1)) and the total and relative abundances of the three single-cell groups showed marked seasonal patterns. Distinct depth distributions during summer stratification and different relations with temperature, chlorophyll and bacterial biovolume suggest the existence of different controlling factors on each single-cell property. Pooled leucine incorporation rates were similarly correlated with the abundance of all physiological groups, while specific growth rates were only substantially explained by the percentage of CTC+ cells. However, the ability to reduce CTC proved notably better than the other two single-cell properties at predicting bacterial bulk rates within seasons, suggesting a tight linkage between bacterial individual respiration and biomass production at the community level.

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

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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.

Gold nanoparticles delivery in mammalian live cells: a critical review

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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.

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

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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.

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

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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

Sorting of cells of the same size, shape, and cell cycle stage for a single cell level assay without staining

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Yomo Tetsuya

2006-06-01

Full Text Available Abstract Background Single-cell level studies are being used increasingly to measure cell properties not directly observable in a cell population. High-performance data acquisition systems for such studies have, by necessity, developed in synchrony. However, improvements in sample purification techniques are also required to reveal new phenomena. Here we assessed a cell sorter as a sample-pretreatment tool for a single-cell level assay. A cell sorter is routinely used for selecting one type of cells from a heterogeneous mixture of cells using specific fluorescence labels. In this case, we wanted to select cells of exactly the same size, shape, and cell-cycle stage from a population, without using a specific fluorescence label. Results We used four light scatter parameters: the peak height and area of the forward scatter (FSheight and FSarea and side scatter (SSheight and SSarea. The rat pheochromocytoma PC12 cell line, a neuronal cell line, was used for all experiments. The living cells concentrated in the high FSarea and middle SSheight/SSarea fractions. Single cells without cell clumps were concentrated in the low SS and middle FS fractions, and in the higher FSheight/FSarea and SSheight/SSarea fractions. The cell populations from these viable, single-cell-rich fractions were divided into twelve subfractions based on their FSarea-SSarea profiles, for more detailed analysis. We found that SSarea was proportional to the cell volume and the FSarea correlated with cell roundness and elongation, as well as with the level of DNA in the cell. To test the method and to characterize the basic properties of the isolated single cells, sorted cells were cultured in separate wells. The cells in all subfractions survived, proliferated and differentiated normally, suggesting that there was no serious damage. The smallest, roundest, and smoothest cells had the highest viability. There was no correlation between proliferation and differentiation. NGF increases

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

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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.

Single Cell Oncogenesis

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Lu, Xin

It is believed that cancer originates from a single cell that has gone through generations of evolution of genetic and epigenetic changes that associate with the hallmarks of cancer. In some cancers such as various types of leukemia, cancer is clonal. Yet in other cancers like glioblastoma (GBM), there is tremendous tumor heterogeneity that is likely to be caused by simultaneous evolution of multiple subclones within the same tissue. It is obvious that understanding how a single cell develops into a clonal tumor upon genetic alterations, at molecular and cellular levels, holds the key to the real appreciation of tumor etiology and ultimate solution for therapeutics. Surprisingly very little is known about the process of spontaneous tumorigenesis from single cells in human or vertebrate animal models. The main reason is the lack of technology to track the natural process of single cell changes from a homeostatic state to a progressively cancerous state. Recently, we developed a patented compound, photoactivatable (''caged'') tamoxifen analogue 4-OHC and associated technique called optochemogenetic switch (OCG switch), which we believe opens the opportunity to address this urgent biological as well as clinical question about cancer. We propose to combine OCG switch with genetically engineered mouse models of head and neck squamous cell carcinoma and high grade astrocytoma (including GBM) to study how single cells, when transformed through acute loss of tumor suppressor genes PTEN and TP53 and gain of oncogenic KRAS, can develop into tumor colonies with cellular and molecular heterogeneity in these tissues. The abstract is for my invited talk in session ``Beyond Darwin: Evolution in Single Cells'' 3/18/2016 11:15 AM.

Role of ICAM-1 polymorphisms (G241R, K469E) in mediating its single-molecule binding ability: Atomic force microscopy measurements on living cells

Energy Technology Data Exchange (ETDEWEB)

Bai, Rui [Chinese (301) General Hospital, 28 Fuxing Road, Haidian District, Beijing 100853 (China); Yi, Shaoqiong [Beijing Institute of Biotechnology, 20 Dongdajie, Fengtai, Beijing 100071 (China); Zhang, Xuejie [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry Chinese Academy of Sciences, 2 Zhongguancun North 1st Street, Beijing 100190 (China); Liu, Huiliang, E-mail: lhl518@vip.sina.com [Department of Cardiology, The General Hospital of Chinese People’s Armed Police Forces, Beijing 100039 (China); Fang, Xiaohong, E-mail: xfang@iccas.ac.cn [Beijing National Laboratory for Molecular Sciences, Key Laboratory of Molecular Nanostructure and Nanotechnology, Institute of Chemistry Chinese Academy of Sciences, 2 Zhongguancun North 1st Street, Beijing 100190 (China)

2014-06-13

Highlights: • We evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations. • AFM was used to measure single-molecule binding ability on living cells. • The SNP of ICAM-1 may induce changes in expressions rather than single-molecule binding ability. - Abstract: Atherosclerosis (As) is characterized by chronic inflammation and is a major cause of human mortality. ICAM-1-mediated adhesion of leukocytes in vessel walls plays an important role in the pathogenesis of atherosclerosis. Two single nucleotide polymorphisms (SNPs) of human intercellular adhesion molecule-1 (ICAM-1), G241R and K469E, are associated with a number of inflammatory diseases. SNP induced changes in ICAM-1 function rely not only on the expression level but also on the single-molecule binding ability which may be affected by single molecule conformation variations such as protein splicing and folding. Previous studies have shown associations between G241R/K469E polymorphisms and ICAM-1 gene expression. Nevertheless, few studies have been done that focus on the single-molecule forces of the above SNPs and their ligands. In the current study, we evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations – GK (G241/K469), GE (G241/E469), RK (R241/K469) and RE (R241/E469). No difference in adhesion ability was observed via cell adhesion assay or atomic force microscopy (AFM) measurement when comparing the GK, GE, RK, or RE genotypes of ICAM-1 to each other. On the other hand, flow cytometry suggested that there was significantly higher expression of GE genotype of ICAM-1 on transfected CHO cells. Thus, we concluded that genetic susceptibility to diseases related to ICAM-1 polymorphisms, G241R or K469E, might be due to the different expressions of ICAM-1 variants rather than to the single-molecule binding ability of ICAM-1.

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)

Single Cell Genomics and Transcriptomics for Unicellular Eukaryotes

Energy Technology Data Exchange (ETDEWEB)

Ciobanu, Doina; Clum, Alicia; Singh, Vasanth; Salamov, Asaf; Han, James; Copeland, Alex; Grigoriev, Igor; James, Timothy; Singer, Steven; Woyke, Tanja; Malmstrom, Rex; Cheng, Jan-Fang

2014-03-14

Despite their small size, unicellular eukaryotes have complex genomes with a high degree of plasticity that allow them to adapt quickly to environmental changes. Unicellular eukaryotes live with prokaryotes and higher eukaryotes, frequently in symbiotic or parasitic niches. To this day their contribution to the dynamics of the environmental communities remains to be understood. Unfortunately, the vast majority of eukaryotic microorganisms are either uncultured or unculturable, making genome sequencing impossible using traditional approaches. We have developed an approach to isolate unicellular eukaryotes of interest from environmental samples, and to sequence and analyze their genomes and transcriptomes. We have tested our methods with six species: an uncharacterized protist from cellulose-enriched compost identified as Platyophrya, a close relative of P. vorax; the fungus Metschnikowia bicuspidate, a parasite of water flea Daphnia; the mycoparasitic fungi Piptocephalis cylindrospora, a parasite of Cokeromyces and Mucor; Caulochytrium protosteloides, a parasite of Sordaria; Rozella allomycis, a parasite of the water mold Allomyces; and the microalgae Chlamydomonas reinhardtii. Here, we present the four components of our approach: pre-sequencing methods, sequence analysis for single cell genome assembly, sequence analysis of single cell transcriptomes, and genome annotation. This technology has the potential to uncover the complexity of single cell eukaryotes and their role in the environmental samples.

Single-cell sequencing in stem cell biology.

Science.gov (United States)

Wen, Lu; Tang, Fuchou

2016-04-15

Cell-to-cell variation and heterogeneity are fundamental and intrinsic characteristics of stem cell populations, but these differences are masked when bulk cells are used for omic analysis. Single-cell sequencing technologies serve as powerful tools to dissect cellular heterogeneity comprehensively and to identify distinct phenotypic cell types, even within a 'homogeneous' stem cell population. These technologies, including single-cell genome, epigenome, and transcriptome sequencing technologies, have been developing rapidly in recent years. The application of these methods to different types of stem cells, including pluripotent stem cells and tissue-specific stem cells, has led to exciting new findings in the stem cell field. In this review, we discuss the recent progress as well as future perspectives in the methodologies and applications of single-cell omic sequencing technologies.

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.

Interactions between semiconductor nanowires and living cells.

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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.

Mass Cytometry for Detection of Silver at the Bacterial Single Cell Level

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Yuting Guo

2017-07-01

Full Text Available Background: Mass cytometry (Cytometry by Time of Flight, CyTOF allows single-cell characterization on the basis of specific metal-based cell markers. In addition, other metals in the mass range such as silver can be detected per cell. Bacteria are known to be sensible to silver and a protocol was developed to measure both the number of affected cells per population and the quantities of silver per cell.Methods: For mass cytometry ruthenium red was used as a marker for all cells of a population while parallel application of cisplatin discriminated live from dead cells. Silver quantities per cell and frequencies of silver containing cells in a population were measured by mass cytometry. In addition, live/dead subpopulations were analyzed by flow cytometry and distinguished by cell sorting based on ruthenium red and propidium iodide double staining. Verification of the cells’ silver load was performed on the bulk level by using ICP-MS in combination with cell sorting. The protocol was developed by conveying both, fast and non-growing Pseudomonas putida cells as test organisms.Results: A workflow for labeling bacteria in order to be analyzed by mass cytometry was developed. Three different parameters were tested: ruthenium red provided counts for all bacterial cells in a population while consecutively applied cisplatin marked the frequency of dead cells. Apparent population heterogeneity was detected by different frequencies of silver containing cells. Silver quantities per cell were also well measurable. Generally, AgNP-10 treatment caused higher frequencies of dead cells, higher frequencies of silver containing cells and higher per-cell silver quantities. Due to an assumed chemical equilibrium of free and bound silver ions live and dead cells were associated with silver in equal quantities and this preferably during exponential growth. With ICP-MS up to 1.5 fg silver per bacterial cell were detected.Conclusion: An effective mass cytometry

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.

A Label-Free Microfluidic Biosensor for Activity Detection of Single Microalgae Cells Based on Chlorophyll Fluorescence

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Junsheng Wang

2013-11-01

Full Text Available Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis.

A Label-Free Microfluidic Biosensor for Activity Detection of Single Microalgae Cells Based on Chlorophyll Fluorescence

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Wang, Junsheng; Sun, Jinyang; Song, Yongxin; Xu, Yongyi; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

2013-01-01

Detection of living microalgae cells is very important for ballast water treatment and analysis. Chlorophyll fluorescence is an indicator of photosynthetic activity and hence the living status of plant cells. In this paper, we developed a novel microfluidic biosensor system that can quickly and accurately detect the viability of single microalgae cells based on chlorophyll fluorescence. The system is composed of a laser diode as an excitation light source, a photodiode detector, a signal analysis circuit, and a microfluidic chip as a microalgae cell transportation platform. To demonstrate the utility of this system, six different living and dead algae samples (Karenia mikimotoi Hansen, Chlorella vulgaris, Nitzschia closterium, Platymonas subcordiformis, Pyramidomonas delicatula and Dunaliella salina) were tested. The developed biosensor can distinguish clearly between the living microalgae cells and the dead microalgae cells. The smallest microalgae cells that can be detected by using this biosensor are 3 μm ones. Even smaller microalgae cells could be detected by increasing the excitation light power. The developed microfluidic biosensor has great potential for in situ ballast water analysis. PMID:24287532

Single Nanowire Probe for Single Cell Endoscopy and Sensing

Science.gov (United States)

Yan, Ruoxue

The ability to manipulate light in subwavelength photonic and plasmonic structures has shown great potentials in revolutionizing how information is generated, transformed and processed. Chemically synthesized nanowires, in particular, offers a unique toolbox not only for highly compact and integrated photonic modules and devices, including coherent and incoherent light sources, waveguides, photodetectors and photovoltaics, but also for new types of nanoscopic bio-probes for spot cargo delivery and in-situ single cell endoscopy and sensing. Such nanowire probes would enable us to carry out intracellular imaging and probing with high spatial resolution, monitor in-vivo biological processes within single living cells and greatly improve our fundamental understanding of cell functions, intracellular physiological processes, and cellular signal pathways. My work is aimed at developing a material and instrumental platform for such single nanowire probe. Successful optical integration of Ag nanowire plasmonic waveguides, which offers deep subwavelength mode confinement, and conventional photonic waveguides was demonstrated on a single nanowire level. The highest plasmonic-photonic coupling efficiency coupling was found at small coupling angles and low input frequencies. The frequency dependent propagation loss was observed in Ag nanowire and was confirmed by quantitative measurement and in agreement with theoretical expectations. Rational integration of dielectric and Ag nanowire waveguide components into hybrid optical-plasmonic routing devices has been demonstrated. This capability is essential for incorporating sub-100nm Ag nanowire waveguides into optical fiber based nanoprobes for single cell endoscopy. The nanoprobe system based on single nanowire waveguides was demonstrated by optically coupling semiconductor or metal nanowire with an optical fiber with tapered tip. This nanoprobe design requires minimal instrumentation which makes it cost efficient and readily

4Pi-confocal microscopy of live cells

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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.

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

Single-photon ultrashort-lived radionuclides: symposium proceedings

Energy Technology Data Exchange (ETDEWEB)

Paras, P.; Thiessen, J.W. (eds.)

1985-01-01

The purpose was to define the current role and state-of-the-art regarding the development, clinical applications, and usefulness of generator-produced single-photon ultrashort-lived radionuclides (SPUSLR's) and to predict their future impact on medicine. Special emphasis was placed on the generator production of iridium-191, gold-195, and krypton-81. This report contains expanded summaries of the included papers. (ACR)

Functional magnetic resonance microscopy at single-cell resolution in Aplysia californica

Science.gov (United States)

Radecki, Guillaume; Nargeot, Romuald; Jelescu, Ileana Ozana; Le Bihan, Denis; Ciobanu, Luisa

2014-01-01

In this work, we show the feasibility of performing functional MRI studies with single-cell resolution. At ultrahigh magnetic field, manganese-enhanced magnetic resonance microscopy allows the identification of most motor neurons in the buccal network of Aplysia at low, nontoxic Mn2+ concentrations. We establish that Mn2+ accumulates intracellularly on injection into the living Aplysia and that its concentration increases when the animals are presented with a sensory stimulus. We also show that we can distinguish between neuronal activities elicited by different types of stimuli. This method opens up a new avenue into probing the functional organization and plasticity of neuronal networks involved in goal-directed behaviors with single-cell resolution. PMID:24872449

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...

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.

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.

A New Genetically Encoded Single-Chain Biosensor for Cdc42 Based on FRET, Useful for Live-Cell Imaging

Science.gov (United States)

Cox, Dianne; Hodgson, Louis

2014-01-01

Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized. PMID:24798463

Microfluidics for single cell analysis

DEFF Research Database (Denmark)

Jensen, Marie Pødenphant

Isolation and manipulation of single cells have gained an increasing interest from researchers because of the heterogeneity of cells from the same cell culture. Single cell analysis can ensure a better understanding of differences between individual cells and potentially solve a variety of clinical...... problems. In this thesis lab on a chip systems for rare single cell analysis are investigated. The focus was to develop a commercial, disposable device for circulating tumour cell (CTC) analysis. Such a device must be able to separate rare cells from blood samples and subsequently capture the specific...... cells, and simultaneously be fabricated and operated at low costs and be user-friendly. These challenges were addressed through development of two microfluidic devices, one for rare cell isolation based on pinched flow fractionation (PFF) and one for single cell capture based on hydrodynamic trapping...

Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells.

Science.gov (United States)

Pi, Jiang; Jin, Hua; Jiang, Jinhuan; Yang, Fen; Cai, Huaihong; Yang, Peihui; Cai, Jiye; Chen, Zheng W

2017-05-01

As the active anticancer component of Rabdosia Rubescens, oridonin has been proved to show strong anticancer activity in cancer cells, which is also found to be closely related to its specific inhibition effects on the EGFR tyrosine kinase activity. In this study, atomic force microscopy based single molecule force spectroscopy (AFM-SMFS) was used for real-time and in-situ detection of EGF-EGFR interactions in living esophageal cancer KYSE-150 cells to evaluate the anticancer activity of oridonin for the first time. Oridonin was found to induce apoptosis and also reduce EGFR expression in KYSE-150 cells. AFM-SMFS results demonstrated that oridonin could inhibit the binding between EGF and EGFR in KYSE-150 cells by decreasing the unbinding force and binding probability for EGF-EGFR complexes, which was further proved to be closely associated with the intracellular ROS level. More precise mechanism studies based on AFM-SMFS demonstrated that oridonin treatment could decrease the energy barrier width, increase the dissociation off rate constant and decrease the activation energy of EGF-EGFR complexes in ROS dependent way, suggesting oridonin as a strong anticancer agent targeting EGF-EGFR interactions in cancer cells through ROS dependent mechanism. Our results not only suggested oridonin as a strong anticancer agent targeting EGF-EGFR interactions in ROS dependent mechanism, but also highlighted AFM-SMFS as a powerful technique for pharmacodynamic studies by detecting ligand-receptor interactions, which was also expected to be developed into a promising tool for the screening and mechanism studies of drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

An Optogenetic Platform for Real-Time, Single-Cell Interrogation of Stochastic Transcriptional Regulation.

Science.gov (United States)

Rullan, Marc; Benzinger, Dirk; Schmidt, Gregor W; Milias-Argeitis, Andreas; Khammash, Mustafa

2018-05-17

Transcription is a highly regulated and inherently stochastic process. The complexity of signal transduction and gene regulation makes it challenging to analyze how the dynamic activity of transcriptional regulators affects stochastic transcription. By combining a fast-acting, photo-regulatable transcription factor with nascent RNA quantification in live cells and an experimental setup for precise spatiotemporal delivery of light inputs, we constructed a platform for the real-time, single-cell interrogation of transcription in Saccharomyces cerevisiae. We show that transcriptional activation and deactivation are fast and memoryless. By analyzing the temporal activity of individual cells, we found that transcription occurs in bursts, whose duration and timing are modulated by transcription factor activity. Using our platform, we regulated transcription via light-driven feedback loops at the single-cell level. Feedback markedly reduced cell-to-cell variability and led to qualitative differences in cellular transcriptional dynamics. Our platform establishes a flexible method for studying transcriptional dynamics in single cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Development of the IFJ single ion hit facility for cells irradiation

International Nuclear Information System (INIS)

Veselov, O.; Polak, W.; Ugenskiene, R.; Hajduk, R.; Lebed, K.; Lekki, J.; Horwacik, T.; Dutkiewicz, E.M.; Maranda, S.; Pieprzyca, T.; Sarnecki, C.; Stachura, Z.; Szklarz, Z.; Styczen, J.

2005-12-01

In recent years a single ion hit facility (SIHF) has been constructed at the IFJ ion microprobe. The setup is used for the precise irradiations of living cells by a controlled number of ions. The facility allows investigations in various aspects of biomedical research, such as adaptive response, bystander effect, inverse dose-rate effect, low-dose hypersensitivity, etc. Those investigations have two very important requirements: (i) cells must be examined in their natural state and environment, i.e. without previously being killed, and preferentially, neither fixed nor stained, and (ii) a possibility of automatic irradiation of large number of cells with a computer recognition of their positions must be provided. This work presents some of the crucial features of the off-line and on-line optical systems, including self-developed software responsible for the automatic cell recognition. We also show several tests carried out to determine the efficiency of the whole setup and some segments. In conclusion, the results of our first irradiation measurements performed with living cells are demonstrated. (author)

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.

Robust organelle size extractions from elastic scattering measurements of single cells (Conference Presentation)

Science.gov (United States)

Cannaday, Ashley E.; Draham, Robert; Berger, Andrew J.

2016-04-01

The goal of this project is to estimate non-nuclear organelle size distributions in single cells by measuring angular scattering patterns and fitting them with Mie theory. Simulations have indicated that the large relative size distribution of organelles (mean:width≈2) leads to unstable Mie fits unless scattering is collected at polar angles less than 20 degrees. Our optical system has therefore been modified to collect angles down to 10 degrees. Initial validations will be performed on polystyrene bead populations whose size distributions resemble those of cell organelles. Unlike with the narrow bead distributions that are often used for calibration, we expect to see an order-of-magnitude improvement in the stability of the size estimates as the minimum angle decreases from 20 to 10 degrees. Scattering patterns will then be acquired and analyzed from single cells (EMT6 mouse cancer cells), both fixed and live, at multiple time points. Fixed cells, with no changes in organelle sizes over time, will be measured to determine the fluctuation level in estimated size distribution due to measurement imperfections alone. Subsequent measurements on live cells will determine whether there is a higher level of fluctuation that could be attributed to dynamic changes in organelle size. Studies on unperturbed cells are precursors to ones in which the effects of exogenous agents are monitored over time.

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.

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.

Single Cell Isolation and Analysis

Directory of Open Access Journals (Sweden)

Ping Hu

2016-10-01

Full Text Available Increasing evidence shows that the heterogeneity of individual cells within a genetically identical population can be critical to their peculiar function and fate. Conventional cell based assays mainly analysis the average responses from a population cells, while the difference within individual cells may often be masked. The cell size, RNA transcripts and protein expression level are quite different within individual cells and these variations are key point to answer the problems in cancer, neurobiology, stem cell biology, immunology and developmental biology. To better understand the cell-to-cell variations, the single cell analysis can provide much more detailed information which may be helpful for therapeutic decisions in an increasingly personalized medicine. In this review, we will focus on the recent development in single cell analysis, including methods used in single cell isolation, analysis and some application examples. The review provides the historical background to single cell analysis, discusses limitations, and current and future possibilities in this exciting field of research.

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.

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.

Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system

International Nuclear Information System (INIS)

Matsumae, Yoshiharu; Takahashi, Yasufumi; Ino, Kosuke; Shiku, Hitoshi; Matsue, Tomokazu

2014-01-01

Graphical abstract: NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells were evaluated by using the menadione–ferrocyanide double mediator system combined with scanning electrochemical microscopy (SECM). - Highlights: • NAD(P)H:quinone oxidoreductase activity of single cells were evaluated with SECM. • Fe(CN) 6 3− /menadione concentrations were optimized for long-term SECM monitoring. • Menadione affect the intracellular levels of reactive oxygen species and GSH. • At 100 μM menadione, the Fe(CN) 6 3− generation rate decreased rapidly within 30 min. - Abstract: We evaluated the intracellular NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione–ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 μM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 μM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system

Electrochemical monitoring of intracellular enzyme activity of single living mammalian cells by using a double-mediator system

Energy Technology Data Exchange (ETDEWEB)

Matsumae, Yoshiharu [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Takahashi, Yasufumi [Advanced Institute for Materials Research, Tohoku University, Katahira 2-1-1, Aoba, Sendai 980-8577 (Japan); Ino, Kosuke [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Shiku, Hitoshi, E-mail: shiku@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Matsue, Tomokazu, E-mail: matsue@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, Aramaki 6-6-11-605, Aoba, Sendai 980-8579 (Japan); Advanced Institute for Materials Research, Tohoku University, Katahira 2-1-1, Aoba, Sendai 980-8577 (Japan)

2014-09-09

Graphical abstract: NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells were evaluated by using the menadione–ferrocyanide double mediator system combined with scanning electrochemical microscopy (SECM). - Highlights: • NAD(P)H:quinone oxidoreductase activity of single cells were evaluated with SECM. • Fe(CN){sub 6}{sup 3−}/menadione concentrations were optimized for long-term SECM monitoring. • Menadione affect the intracellular levels of reactive oxygen species and GSH. • At 100 μM menadione, the Fe(CN){sub 6}{sup 3−} generation rate decreased rapidly within 30 min. - Abstract: We evaluated the intracellular NAD(P)H:quinone oxidoreductase (NQO) activity of single HeLa cells by using the menadione–ferrocyanide double-mediator system combined with scanning electrochemical microscopy (SECM). The double-mediator system was used to amplify the current response from the intracellular NQO activity and to reduce menadione-induced cell damage. The electron shuttle between the electrode and menadione was mediated by the ferrocyanide/ferricyanide redox couple. Generation of ferrocyanide was observed immediately after the addition of a lower concentration (10 μM) of menadione. The ferrocyanide generation rate was constant for 120 min. At a higher menadione concentration (100 μM), the ferrocyanide generation rate decreased within 30 min because of the cytotoxic effect of menadione. We also investigated the relationship between intracellular reactive oxygen species or glutathione levels and exposure to different menadione concentrations to determine the optimal condition for SECM with minimal invasiveness. The present study clearly demonstrates that SECM is useful for the analysis of intracellular enzymatic activities in single cells with a double-mediator system.

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

Live Imaging of Cellular Internalization of Single Colloidal Particle by Combined Label-Free and Fluorescence Total Internal Reflection Microscopy.

Science.gov (United States)

Byrne, Gerard D; Vllasaliu, Driton; Falcone, Franco H; Somekh, Michael G; Stolnik, Snjezana

2015-11-02

In this work we utilize the combination of label-free total internal reflection microscopy and total internal reflectance fluorescence (TIRM/TIRF) microscopy to achieve a simultaneous, live imaging of single, label-free colloidal particle endocytosis by individual cells. The TIRM arm of the microscope enables label free imaging of the colloid and cell membrane features, while the TIRF arm images the dynamics of fluorescent-labeled clathrin (protein involved in endocytosis via clathrin pathway), expressed in transfected 3T3 fibroblasts cells. Using a model polymeric colloid and cells with a fluorescently tagged clathrin endocytosis pathway, we demonstrate that wide field TIRM/TIRF coimaging enables live visualization of the process of colloidal particle interaction with the labeled cell structure, which is valuable for discerning the membrane events and route of colloid internalization by the cell. We further show that 500 nm in diameter model polystyrene colloid associates with clathrin, prior to and during its cellular internalization. This association is not apparent with larger, 1 μm in diameter colloids, indicating an upper particle size limit for clathrin-mediated endocytosis.

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

Science.gov (United States)

MacIntyre, Hugh L; Cullen, John J

2016-08-01

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

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

Lateral motion and bending of microtubules studied with a new single-filament tracking routine in living cells.

Science.gov (United States)

Pallavicini, Carla; Levi, Valeria; Wetzler, Diana E; Angiolini, Juan F; Benseñor, Lorena; Despósito, Marcelo A; Bruno, Luciana

2014-06-17

The cytoskeleton is involved in numerous cellular processes such as migration, division, and contraction and provides the tracks for transport driven by molecular motors. Therefore, it is very important to quantify the mechanical behavior of the cytoskeletal filaments to get a better insight into cell mechanics and organization. It has been demonstrated that relevant mechanical properties of microtubules can be extracted from the analysis of their motion and shape fluctuations. However, tracking individual filaments in living cells is extremely complex due, for example, to the high and heterogeneous background. We introduce a believed new tracking algorithm that allows recovering the coordinates of fluorescent microtubules with ∼9 nm precision in in vitro conditions. To illustrate potential applications of this algorithm, we studied the curvature distributions of fluorescent microtubules in living cells. By performing a Fourier analysis of the microtubule shapes, we found that the curvatures followed a thermal-like distribution as previously reported with an effective persistence length of ∼20 μm, a value significantly smaller than that measured in vitro. We also verified that the microtubule-associated protein XTP or the depolymerization of the actin network do not affect this value; however, the disruption of intermediate filaments decreased the persistence length. Also, we recovered trajectories of microtubule segments in actin or intermediate filament-depleted cells, and observed a significant increase of their motion with respect to untreated cells showing that these filaments contribute to the overall organization of the microtubule network. Moreover, the analysis of trajectories of microtubule segments in untreated cells showed that these filaments presented a slower but more directional motion in the cortex with respect to the perinuclear region, and suggests that the tracking routine would allow mapping the microtubule dynamical organization in cells

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...

Live cell imaging at the Munich ion microbeam SNAKE – a status report

International Nuclear Information System (INIS)

Drexler, Guido A; Siebenwirth, Christian; Drexler, Sophie E; Girst, Stefanie; Greubel, Christoph; Dollinger, Günther; Friedl, Anna A

2015-01-01

Ion microbeams are important tools in radiobiological research. Still, the worldwide number of ion microbeam facilities where biological experiments can be performed is limited. Even fewer facilities combine ion microirradiation with live-cell imaging to allow microscopic observation of cellular response reactions starting very fast after irradiation and continuing for many hours. At SNAKE, the ion microbeam facility at the Munich 14 MV tandem accelerator, a large variety of biological experiments are performed on a regular basis. Here, recent developments and ongoing research projects at the ion microbeam SNAKE are presented with specific emphasis on live-cell imaging experiments. An overview of the technical details of the setup is given, including examples of suitable biological samples. By ion beam focusing to submicrometer beam spot size and single ion detection it is possible to target subcellular structures with defined numbers of ions. Focusing of high numbers of ions to single spots allows studying the influence of high local damage density on recruitment of damage response proteins. The online version of this article (doi:10.1186/s13014-015-0350-7) contains supplementary material, which is available to authorized users

Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae.

Science.gov (United States)

Higuchi-Sanabria, Ryo; Garcia, Enrique J; Tomoiaga, Delia; Munteanu, Emilia L; Feinstein, Paul; Pon, Liza A

2016-01-01

Saccharomyces cerevisiae are widely used for imaging fluorescently tagged protein fusions. Fluorescent proteins can easily be inserted into yeast genes at their chromosomal locus, by homologous recombination, for expression of tagged proteins at endogenous levels. This is especially useful for incorporation of multiple fluorescent protein fusions into a single strain, which can be challenging in organisms where genetic manipulation is more complex. However, the availability of optimal fluorescent protein combinations for 3-color imaging is limited. Here, we have characterized a combination of fluorescent proteins, mTFP1/mCitrine/mCherry for multicolor live cell imaging in S. cerevisiae. This combination can be used with conventional blue dyes, such as DAPI, for potential four-color live cell imaging.

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.

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.

Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

Science.gov (United States)

Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

2016-03-01

Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

Technologies for Single-Cell Isolation

Science.gov (United States)

Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

2015-01-01

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field. PMID:26213926

Technologies for Single-Cell Isolation

Directory of Open Access Journals (Sweden)

Andre Gross

2015-07-01

Full Text Available The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting respectively Flow cytometry (33% usage, laser microdissection (17%, manual cell picking (17%, random seeding/dilution (15%, and microfluidics/lab-on-a-chip devices (12% are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

Technologies for Single-Cell Isolation.

Science.gov (United States)

Gross, Andre; Schoendube, Jonas; Zimmermann, Stefan; Steeb, Maximilian; Zengerle, Roland; Koltay, Peter

2015-07-24

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

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.

Three-dimensional single-particle tracking in live cells: news from the third dimension

International Nuclear Information System (INIS)

Dupont, A; Wehnekamp, F; Katayama, Y; Lamb, D C; Gorelashvili, M; Schüller, V; Arcizet, D; Heinrich, D

2013-01-01

Single-particle tracking (SPT) is of growing importance in the biophysical community. It is used to investigate processes such as drug and gene delivery, viral uptake, intracellular trafficking or membrane-bound protein mobility. Traditionally, SPT is performed in two dimensions (2D) because of its technical simplicity. However, life occurs in three dimensions (3D) and many methods have been recently developed to track particles in 3D. Now, is the third dimension worth the effort? Here we investigate the differences between the 2D and 3D analyses of intracellular transport with the 3D development of a time-resolved mean square displacement (MSD) analysis introduced previously. The 3D trajectories, and the 2D projections, of fluorescent nanoparticles were obtained with an orbital tracking microscope in two different cell types: in Dictyostelium discoideum ameba and in adherent, more flattened HuH-7 human cells. As expected from the different 3D organization of both cells’ cytoskeletons, a third of the active transport was lost upon projection in the ameba whereas the identification of the active phases was barely affected in the HuH-7 cells. In both cell types, we found intracellular diffusion to be anisotropic and the diffusion coefficient values derived from the 2D analysis were therefore biased. (paper)

Everyday life and occupational deprivation in single migrant mothers living in Denmark

DEFF Research Database (Denmark)

Kielsgaard, Kamilla; Kristensen, Hanne Kaae; Nielsen, Dorthe Susanne

2018-01-01

/objectives: To explore how single migrant mothers experience their living conditions and the significance of those conditions on their exclusion from everyday life occupations. Material and methods: In-depth interviews were used to find how occupational deprivation manifests in the everyday lives of three women. Based......ABSTRACT Background: Socio-economically disadvantaged single migrant mothers in Denmark risk poor health and social marginalisation, which affects participation in relevant occupations. Literature focusing on occupational deprivation in vulnerable groups such as migrants is sparse. Aim...... on Ricoeur’s theory of interpretation, data were analysed and the meaning structures, in the form of three themes, were identified. Results: The societal and individual conditions of women’s everyday lives interact in a complex interplay, where immigration, illness and divorce, in particular, deprive...

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.

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.

Elemental analysis of single phytoplankton cells using the Lund nuclear microprobe

International Nuclear Information System (INIS)

Pallon, Jan; Elfman, Mikael; Kristiansson, Per; Malmqvist, Klas; Graneli, Edna; Sellborn, Anders; Karlsson, Chatarina

1999-01-01

The occurrence of annual marine phytoplankton blooms is becoming a global problem. In Europe, the NUTOX project supported by the EC investigates if unbalanced nutrient compositions in the water promote the dominance of harmful phytoplankton species. One of the tasks is the determination of the elemental composition of single phytoplankton cells. This is carried out using the Lund Nuclear Microprobe with a special focus on C, N, P and K. The overall aim is to understand the mechanism leading to toxin production, model it and eventually propose a counteracting method. The preparative method, used to isolate single living cells while reducing their salt environment, is an important part of the analytical procedure. A comparison of light element detection using backscattering from protons and nuclear reaction analysis using deuterons is made

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

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

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…

Single living predicts a higher mortality in both women and men with chronic heart failure

DEFF Research Database (Denmark)

Mard, Shan; Nielsen, Finn Erland

2016-01-01

INTRODUCTION: We examined the impact of single living on all-cause mortality in patients with chronic heart failure and determined if this association was modified by gender. METHODS: This historical cohort study included 637 patients who were admitted to the Department of Cardiology, Herlev Hosp......, the risk of death did not differ among single-living women and men. CONCLUSION: Single living is a prognostic determinant of all-cause mortality in men and women with chronic heart failure. FUNDING: none. TRIAL REGISTRATION: not relevant.......INTRODUCTION: We examined the impact of single living on all-cause mortality in patients with chronic heart failure and determined if this association was modified by gender. METHODS: This historical cohort study included 637 patients who were admitted to the Department of Cardiology, Herlev...... for confounding factors. RESULTS: The median follow-up time was 2.8 years. A total of 323 (50.7%) patients died during the follow-up period. After adjustment for confounding factors, risk of death was associated with being single (HR = 1.53 (95% confidence interval: 1.19-1.96)). In a gender-stratified analysis...

Quantification of the number of EP3 receptors on a living CHO cell surface by the AFM

International Nuclear Information System (INIS)

Kim, Hyonchol; Arakawa, Hideo; Hatae, Noriyuki; Sugimoto, Yukihiko; Matsumoto, Osamu; Osada, Toshiya; Ichikawa, Atsushi; Ikai, Atsushi

2006-01-01

The distribution of EP3 receptors on a living cell surface was quantitatively studied by atomic force microscopy (AFM). Green fluorescent protein (GFP) was introduced to the extracellular region of the EP3 receptor on a CHO cell. A microbead was used as a probe to ensure certain contact area, whose surface was coated with anti-GFP antibody. The interactions between the antibodies and GFP molecules on the cell surface were recorded to observe the distribution of the receptors. The result indicated that EP3 receptors were distributed on the CHO cell surface not uniformly but in small patches coincident with immunohistochemical observation. Repeated measurements on the same area of cell surface gave confirmation that it was unlikely that the receptors were extracted from the cell membrane during the experiments. The measurement of single molecular interaction between GFP and the anti-GFP antibody was succeeded on the cell surface using compression-free force spectroscopy. The value of separation work required to break a single molecular pair was estimated to be about 1.5x10 -18 J. The number of EP3 receptor on the CHO cell surface was estimated using this value to be about 1x10 4 under the assumption that the area of the cell surface was about 5000 μm 2 . These results indicated that the number of receptors on a living cell surface could be quantified through the force measurement by the AFM

Limits to anaerobic energy and cytosolic concentration in the living cell

Science.gov (United States)

Paglietti, A.

2015-11-01

For many physical systems at any given temperature, the set of all states where the system's free energy reaches its largest value can be determined from the system's constitutive equations of internal energy and entropy, once a state of that set is known. Such an approach is fraught with complications when applied to a living cell, because the cell's cytosol contains thousands of solutes, and thus thousands of state variables, which makes determination of its state impractical. We show here that, when looking for the maximum energy that the cytosol can store and release, detailed information on cytosol composition is redundant. Compatibility with cell's life requires that a single variable that represents the overall concentration of cytosol solutes must fall between defined limits, which can be determined by dehydrating and overhydrating the cell to its maximum capacity. The same limits are shown to determine, in particular, the maximum amount of free energy that a cell can supply in fast anaerobic processes, starting from any given initial state. For a typical skeletal muscle in normal physiological conditions this energy, i.e., the maximum anaerobic capacity to do work, is calculated to be about 960 J per kg of muscular mass. Such energy decreases as the overall concentration of solutes in the cytosol is increased. Similar results apply to any kind of cell. They provide an essential tool to understand and control the macroscopic response of single cells and multicellular cellular tissues alike. The applications include sport physiology, cell aging, disease produced cell damage, drug absorption capacity, to mention the most obvious ones.

Single-cell MALDI-MS as an analytical tool for studying intrapopulation metabolic heterogeneity of unicellular organisms.

Science.gov (United States)

Amantonico, Andrea; Urban, Pawel L; Fagerer, Stephan R; Balabin, Roman M; Zenobi, Renato

2010-09-01

Heterogeneity is a characteristic feature of all populations of living organisms. Here we make an attempt to validate a single-cell mass spectrometric method for detection of changes in metabolite levels occurring in populations of unicellular organisms. Selected metabolites involved in central metabolism (ADP, ATP, GTP, and UDP-Glucose) could readily be detected in single cells of Closterium acerosum by means of negative-mode matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS). The analytical capabilities of this approach were characterized using standard compounds. The method was then used to study populations of individual cells with different levels of the chosen metabolites. With principal component analysis and support vector machine algorithms, it was possible to achieve a clear separation of individual C. acerosum cells in different metabolic states. This study demonstrates the suitability of mass spectrometric analysis of metabolites in single cells to measure cell-population heterogeneity.

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.

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

Tilted Light Sheet Microscopy with 3D Point Spread Functions for Single-Molecule Super-Resolution Imaging in Mammalian Cells.

Science.gov (United States)

Gustavsson, Anna-Karin; Petrov, Petar N; Lee, Maurice Y; Shechtman, Yoav; Moerner, W E

2018-02-01

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D super-resolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Tilted light sheet microscopy with 3D point spread functions for single-molecule super-resolution imaging in mammalian cells

Science.gov (United States)

Gustavsson, Anna-Karin; Petrov, Petar N.; Lee, Maurice Y.; Shechtman, Yoav; Moerner, W. E.

2018-02-01

To obtain a complete picture of subcellular nanostructures, cells must be imaged with high resolution in all three dimensions (3D). Here, we present tilted light sheet microscopy with 3D point spread functions (TILT3D), an imaging platform that combines a novel, tilted light sheet illumination strategy with engineered long axial range point spread functions (PSFs) for low-background, 3D super localization of single molecules as well as 3D super-resolution imaging in thick cells. TILT3D is built upon a standard inverted microscope and has minimal custom parts. The axial positions of the single molecules are encoded in the shape of the PSF rather than in the position or thickness of the light sheet, and the light sheet can therefore be formed using simple optics. The result is flexible and user-friendly 3D super-resolution imaging with tens of nm localization precision throughout thick mammalian cells. We validated TILT3D for 3D superresolution imaging in mammalian cells by imaging mitochondria and the full nuclear lamina using the double-helix PSF for single-molecule detection and the recently developed Tetrapod PSF for fiducial bead tracking and live axial drift correction. We envision TILT3D to become an important tool not only for 3D super-resolution imaging, but also for live whole-cell single-particle and single-molecule tracking.

Using measures of single-cell physiology and physiological state to understand organismic aging.

Science.gov (United States)

Mendenhall, Alexander; Driscoll, Monica; Brent, Roger

2016-02-01

Genetically identical organisms in homogeneous environments have different lifespans and healthspans. These differences are often attributed to stochastic events, such as mutations and 'epimutations', changes in DNA methylation and chromatin that change gene function and expression. But work in the last 10 years has revealed differences in lifespan- and health-related phenotypes that are not caused by lasting changes in DNA or identified by modifications to DNA or chromatin. This work has demonstrated persistent differences in single-cell and whole-organism physiological states operationally defined by values of reporter gene signals in living cells. While some single-cell states, for example, responses to oxygen deprivation, were defined previously, others, such as a generally heightened ability to make proteins, were, revealed by direct experiment only recently, and are not well understood. Here, we review technical progress that promises to greatly increase the number of these measurable single-cell physiological variables and measureable states. We discuss concepts that facilitate use of single-cell measurements to provide insight into physiological states and state transitions. We assert that researchers will use this information to relate cell level physiological readouts to whole-organism outcomes, to stratify aging populations into groups based on different physiologies, to define biomarkers predictive of outcomes, and to shed light on the molecular processes that bring about different individual physiologies. For these reasons, quantitative study of single-cell physiological variables and state transitions should provide a valuable complement to genetic and molecular explanations of how organisms age. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

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...

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.

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.

An automated image analysis framework for segmentation and division plane detection of single live Staphylococcus aureus cells which can operate at millisecond sampling time scales using bespoke Slimfield microscopy

Science.gov (United States)

Wollman, Adam J. M.; Miller, Helen; Foster, Simon; Leake, Mark C.

2016-10-01

Staphylococcus aureus is an important pathogen, giving rise to antimicrobial resistance in cell strains such as Methicillin Resistant S. aureus (MRSA). Here we report an image analysis framework for automated detection and image segmentation of cells in S. aureus cell clusters, and explicit identification of their cell division planes. We use a new combination of several existing analytical tools of image analysis to detect cellular and subcellular morphological features relevant to cell division from millisecond time scale sampled images of live pathogens at a detection precision of single molecules. We demonstrate this approach using a fluorescent reporter GFP fused to the protein EzrA that localises to a mid-cell plane during division and is involved in regulation of cell size and division. This image analysis framework presents a valuable platform from which to study candidate new antimicrobials which target the cell division machinery, but may also have more general application in detecting morphologically complex structures of fluorescently labelled proteins present in clusters of other types of cells.

Micromagnetic Cancer Cell Immobilization and Release for Real-Time Single Cell Analysis

Energy Technology Data Exchange (ETDEWEB)

Jaiswal, Devina; Rad, Armin Tahmasbi [Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269 (United States); Nieh, Mu-Ping [Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269 (United States); Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT 06269 (United States); Polymer Program, Institute of Materials Science, University of Connecticut, Storrs, CT 06269 (United States); Claffey, Kevin P. [Department of Cell Biology, University of Connecticut Health Center, Farmington, CT 06030 (United States); Hoshino, Kazunori, E-mail: hoshino@engr.uconn.edu [Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269 (United States)

2017-04-01

Understanding the interaction of live cells with macromolecules is crucial for designing efficient therapies. Considering the functional heterogeneity found in cancer cells, real-time single cell analysis is necessary to characterize responses. In this study, we have designed and fabricated a microfluidic channel with patterned micromagnets which can temporarily immobilize the cells during analysis and release them after measurements. The microchannel is composed of plain coverslip top and bottom panels to facilitate easy microscopic observation and undisturbed application of analytes to the cells. Cells labeled with functionalized magnetic beads were immobilized in the device with an efficiency of 90.8±3.6%. Since the micromagnets are made of soft magnetic material (Ni), they released cells when external magnetic field was turned off from the channel. This allows the reuse of the channel for a new sample. As a model drug analysis, the immobilized breast cancer cells (MCF7) were exposed to fluorescent lipid nanoparticles and association and dissociation were measured through fluorescence analysis. Two concentrations of nanoparticles, 0.06 µg/ml and 0.08 µg/ml were tested and time lapse images were recorded and analyzed. The microfluidic device was able to provide a microenvironment for sample analysis, making it an efficient platform for real-time analysis.

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

Local Nucleosome Dynamics Facilitate Chromatin Accessibility in Living Mammalian Cells

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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.

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)

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.

Fluorescent peptide biosensor for probing the relative abundance of cyclin-dependent kinases in living cells.

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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.

Single charged-particle damage to living cells: a new method based on track-etch detectors

International Nuclear Information System (INIS)

Durante, M.; Grossi, G.F.; Pugliese, M.; Manti, L.; Nappo, M.; Gialanella, G.

1994-01-01

Biological effects of ionizing radiation are usually expressed as a function of the absorbed dose. Low doses of high-LET radiation correspond to one or few particle traversals through the cell. In order to study the biological effectiveness of single charged particles, we have developed a new method based on solid state nuclear track detectors. Cells are seeded on mylar and a LR-115 film is stuck below the mylar base. After irradiation, the LR-115 film is etched and cells observed at a phase contrast microscope connected to a video camera and an image analyzer. In this way, it is possible to measure the number of traversals through the cell nucleus or cytoplasm. Coordinates of each cell on the microscope bench are saved. After incubation for about one week, cells are fixed and stained and the colonies observed at the microscope. The fate of each irradiated cell is therefore correlated to the number of traversals. We have tested this method with two different rodent embryo fibroblast cell lines, C3H 10T1/2 and V79, exposed to 3.2 MeV accelerated α-particles (LET =124 keV/μm). The studied endpoint was cell killing. Preliminary biological results suggest that few α-particle tracks in V79 hamster cells are sufficient to reduce surviving fraction. ((orig.))

Lessons from single-cell transcriptome analysis of oxygen-sensing cells.

Science.gov (United States)

Zhou, Ting; Matsunami, Hiroaki

2018-05-01

The advent of single-cell RNA-sequencing (RNA-Seq) technology has enabled transcriptome profiling of individual cells. Comprehensive gene expression analysis at the single-cell level has proven to be effective in characterizing the most fundamental aspects of cellular function and identity. This unbiased approach is revolutionary for small and/or heterogeneous tissues like oxygen-sensing cells in identifying key molecules. Here, we review the major methods of current single-cell RNA-Seq technology. We discuss how this technology has advanced the understanding of oxygen-sensing glomus cells in the carotid body and helped uncover novel oxygen-sensing cells and mechanisms in the mice olfactory system. We conclude by providing our perspective on future single-cell RNA-Seq research directed at oxygen-sensing cells.

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

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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.

A microfluidic device integrating plasmonic nanodevices for Raman spectroscopy analysis on trapped single living cells

KAUST Repository

Perozziello, Gerardo; Catalano, Rossella; Francardi, Marco; Rondanina, Eliana; Pardeo, Francesca; De Angelis, Francesco De; Malara, Natalia Maria; Candeloro, Patrizio; Morrone, Giovanni; Di Fabrizio, Enzo M.

2013-01-01

In this work we developed a microfluidic device integrating nanoplasmonic devices combined with fluidic trapping regions. The microfuidic traps allow to capture single cells in areas where plasmonic sensors are placed. In this way it is possible to perform Enhanced Raman analysis on the cell membranes. Moreover, by changing direction of the flux it is possible to change the orientation of the cell in the trap, so that it is possible to analyze different points of the membrane of the same cell. We shows an innovative procedure to fabricate and assembly the microfluidic device which combine photolithography, focused ion beam machining, and hybrid bonding between a polymer substrate and lid of Calcium fluoride. This procedure is compatible with the fabrication of the plasmonic sensors in close proximity of the microfluidic traps. Moreover, the use of Calcium fluoride as lid allows full compatibility with Raman measurements producing negligible Raman background signal and avoids Raman artifacts. Finally, we performed Raman analysis on cells to monitor their oxidative stress under particular non physiological conditions. © 2013 Elsevier B.V. All rights reserved.

A microfluidic device integrating plasmonic nanodevices for Raman spectroscopy analysis on trapped single living cells

KAUST Repository

Perozziello, Gerardo

2013-11-01

In this work we developed a microfluidic device integrating nanoplasmonic devices combined with fluidic trapping regions. The microfuidic traps allow to capture single cells in areas where plasmonic sensors are placed. In this way it is possible to perform Enhanced Raman analysis on the cell membranes. Moreover, by changing direction of the flux it is possible to change the orientation of the cell in the trap, so that it is possible to analyze different points of the membrane of the same cell. We shows an innovative procedure to fabricate and assembly the microfluidic device which combine photolithography, focused ion beam machining, and hybrid bonding between a polymer substrate and lid of Calcium fluoride. This procedure is compatible with the fabrication of the plasmonic sensors in close proximity of the microfluidic traps. Moreover, the use of Calcium fluoride as lid allows full compatibility with Raman measurements producing negligible Raman background signal and avoids Raman artifacts. Finally, we performed Raman analysis on cells to monitor their oxidative stress under particular non physiological conditions. © 2013 Elsevier B.V. All rights reserved.

Information management for high content live cell imaging

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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/

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

Science.gov (United States)

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

2012-08-08

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

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

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

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.

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.)

Imaging and quantifying ganglion cells and other transparent neurons in the living human retina.

Science.gov (United States)

Liu, Zhuolin; Kurokawa, Kazuhiro; Zhang, Furu; Lee, John J; Miller, Donald T

2017-11-28

Ganglion cells (GCs) are fundamental to retinal neural circuitry, processing photoreceptor signals for transmission to the brain via their axons. However, much remains unknown about their role in vision and their vulnerability to disease leading to blindness. A major bottleneck has been our inability to observe GCs and their degeneration in the living human eye. Despite two decades of development of optical technologies to image cells in the living human retina, GCs remain elusive due to their high optical translucency. Failure of conventional imaging-using predominately singly scattered light-to reveal GCs has led to a focus on multiply-scattered, fluorescence, two-photon, and phase imaging techniques to enhance GC contrast. Here, we show that singly scattered light actually carries substantial information that reveals GC somas, axons, and other retinal neurons and permits their quantitative analysis. We perform morphometry on GC layer somas, including projection of GCs onto photoreceptors and identification of the primary GC subtypes, even beneath nerve fibers. We obtained singly scattered images by: ( i ) marrying adaptive optics to optical coherence tomography to avoid optical blurring of the eye; ( ii ) performing 3D subcellular image registration to avoid motion blur; and ( iii ) using organelle motility inside somas as an intrinsic contrast agent. Moreover, through-focus imaging offers the potential to spatially map individual GCs to underlying amacrine, bipolar, horizontal, photoreceptor, and retinal pigment epithelium cells, thus exposing the anatomical substrate for neural processing of visual information. This imaging modality is also a tool for improving clinical diagnosis and assessing treatment of retinal disease. Copyright © 2017 the Author(s). Published by PNAS.

Nanoelectronics-biology frontier: From nanoscopic probes for action potential recording in live cells to three-dimensional cyborg tissues

OpenAIRE

Duan, Xiaojie; Fu, Tian-Ming; Liu, Jia; Lieber, Charles M.

2013-01-01

Semiconductor nanowires configured as the active channels of field-effect transistors (FETs) have been used as detectors for high-resolution electrical recording from single live cells, cell networks, tissues and organs. Extracellular measurements with substrate supported silicon nanowire (SiNW) FETs, which have projected active areas orders of magnitude smaller than conventional microfabricated multielectrode arrays (MEAs) and planar FETs, recorded action potential and field potential signa...

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.

Single-cell measurement of red blood cell oxygen affinity.

Science.gov (United States)

Di Caprio, Giuseppe; Stokes, Chris; Higgins, John M; Schonbrun, Ethan

2015-08-11

Oxygen is transported throughout the body by hemoglobin (Hb) in red blood cells (RBCs). Although the oxygen affinity of blood is well-understood and routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of RBC volume and Hb concentration are taken millions of times per day by clinical hematology analyzers, and they are important factors in determining the health of the hematologic system. To better understand the variability and determinants of oxygen affinity on a cellular level, we have developed a system that quantifies the oxygen saturation, cell volume, and Hb concentration for individual RBCs in high throughput. We find that the variability in single-cell saturation peaks at an oxygen partial pressure of 2.9%, which corresponds to the maximum slope of the oxygen-Hb dissociation curve. In addition, single-cell oxygen affinity is positively correlated with Hb concentration but independent of osmolarity, which suggests variation in the Hb to 2,3-diphosphoglycerate (2-3 DPG) ratio on a cellular level. By quantifying the functional behavior of a cellular population, our system adds a dimension to blood cell analysis and other measurements of single-cell variability.

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.

“Sex and the Island”: Lives of Single Women in Prince Edward Island

Directory of Open Access Journals (Sweden)

Kristie Collins

2011-05-01

Full Text Available This article considers the significance attributed to Prince Edward Island in managing a marginalized single female identity, as presented by accounts of thirty never-married and previously-married Island women, aged twenty-seven through sixty-five. As popular media and social narratives overwhelmingly position contemporary single women against an urban backdrop, the question arises as to whether unmarried Island women feel “marooned” in ways their urban counterparts may not. In accordance with feminist aims to produce research for, rather than about, women’s lives, the paper focuses on two themes from fieldwork interviews that were of particular interest to participants. The first theme relates to negotiating female singleness within the Island’s family-centered culture, and the second theme presents participants’ talk around advantages and disadvantages of living in Prince Edward Island, Canada, as single women. The paper concludes with a summary of other findings from the study and suggestions for future research on female singleness and island locales.

Using Force to Probe Single-Molecule Receptor-Cytoskeletal Anchoring Beneath the Surface of a Living Cell

DEFF Research Database (Denmark)

Evans, Evan; Kinoshita, Koji

2007-01-01

-cytoskeletal unbinding increased exponentially with the level of force, suggesting disruption at a site of single-molecule interaction. Since many important enzymes and signaling molecules are closely associated with a membrane receptor-cytoskeletal linkage, pulling on a receptor could alter interactions among its......The ligation of cell surface receptors often communicates a signal that initiates a cytoplasmic chemical cascade to implement an important cell function. Less well understood is how physical stress applied to a cell surface adhesive bond propagates throughout the cytostructure to catalyze...... or trigger important steps in these chemical processes. Probing the nanoscale impact of pulling on cell surface bonds, we discovered that receptors frequently detach prematurely from the interior cytostructure prior to failure of the exterior adhesive bond [Evans, E., Heinrich, V., Leung, A., and Kinoshita...

Single-Cell RNA Sequencing of Glioblastoma Cells.

Science.gov (United States)

Sen, Rajeev; Dolgalev, Igor; Bayin, N Sumru; Heguy, Adriana; Tsirigos, Aris; Placantonakis, Dimitris G

2018-01-01

Single-cell RNA sequencing (sc-RNASeq) is a recently developed technique used to evaluate the transcriptome of individual cells. As opposed to conventional RNASeq in which entire populations are sequenced in bulk, sc-RNASeq can be beneficial when trying to better understand gene expression patterns in markedly heterogeneous populations of cells or when trying to identify transcriptional signatures of rare cells that may be underrepresented when using conventional bulk RNASeq. In this method, we describe the generation and analysis of cDNA libraries from single patient-derived glioblastoma cells using the C1 Fluidigm system. The protocol details the use of the C1 integrated fluidics circuit (IFC) for capturing, imaging and lysing cells; performing reverse transcription; and generating cDNA libraries that are ready for sequencing and analysis.

Single-cell analysis of targeted transcriptome predicts drug sensitivity of single cells within human myeloma tumors.

Science.gov (United States)

Mitra, A K; Mukherjee, U K; Harding, T; Jang, J S; Stessman, H; Li, Y; Abyzov, A; Jen, J; Kumar, S; Rajkumar, V; Van Ness, B

2016-05-01

Multiple myeloma (MM) is characterized by significant genetic diversity at subclonal levels that have a defining role in the heterogeneity of tumor progression, clinical aggressiveness and drug sensitivity. Although genome profiling studies have demonstrated heterogeneity in subclonal architecture that may ultimately lead to relapse, a gene expression-based prediction program that can identify, distinguish and quantify drug response in sub-populations within a bulk population of myeloma cells is lacking. In this study, we performed targeted transcriptome analysis on 528 pre-treatment single cells from 11 myeloma cell lines and 418 single cells from 8 drug-naïve MM patients, followed by intensive bioinformatics and statistical analysis for prediction of proteasome inhibitor sensitivity in individual cells. Using our previously reported drug response gene expression profile signature at the single-cell level, we developed an R Statistical analysis package available at https://github.com/bvnlabSCATTome, SCATTome (single-cell analysis of targeted transcriptome), that restructures the data obtained from Fluidigm single-cell quantitative real-time-PCR analysis run, filters missing data, performs scaling of filtered data, builds classification models and predicts drug response of individual cells based on targeted transcriptome using an assortment of machine learning methods. Application of SCATT should contribute to clinically relevant analysis of intratumor heterogeneity, and better inform drug choices based on subclonal cellular responses.

HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

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Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

2016-03-01

Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

Epigenetics reloaded: the single-cell revolution.

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Bheda, Poonam; Schneider, Robert

2014-11-01

Mechanistically, how epigenetic states are inherited through cellular divisions remains an important open question in the chromatin field and beyond. Defining the heritability of epigenetic states and the underlying chromatin-based mechanisms within a population of cells is complicated due to cell heterogeneity combined with varying levels of stability of these states; thus, efforts must be focused toward single-cell analyses. The approaches presented here constitute the forefront of epigenetics research at the single-cell level using classic and innovative methods to dissect epigenetics mechanisms from the limited material available in a single cell. This review further outlines exciting future avenues of research to address the significance of epigenetic heterogeneity and the contributions of microfluidics technologies to single-cell isolation and analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

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...

Single-living is associated with increased risk of long-term mortality among employed patients with acute myocardial infarction

Directory of Open Access Journals (Sweden)

Finn Erl

2010-05-01

Full Text Available Finn Erland Nielsen, Shan MardDepartment of Cardiology S, Herlev University Hospital, DenmarkObjective: There is conflicting evidence about the impact of social support on adverse outcome after acute myocardial infarction (MI. We examined the relation between single-living and long-term all-cause mortality after MI.Design: A prospective cohort study of 242 employed patients with MI followed up to 16 years after MI.Results: A total of 106 (43.8% patients died during the follow-up. Single-living nearly doubled the risk of death; after adjusting for potential confounding factors, single-living was an independent predictor of death, with a hazard ratio of 2.55 (95% confidence interval: 1.52–4.30. Other predictors of death were diabetes mellitus, atrial fibrillation, age, and ejection fraction less than 35%.Conclusion: Single-living is a prognostic determinant of long-term all-cause mortality after MI.Keywords: acute myocardial infarction, social support, single-living, prognosis.

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

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Cisse, Ibrahim

2014-03-01

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

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

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

2017-02-01

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

A study of the dynamics of PTEN proteins in living cells using in vivo fluorescence correlation spectroscopy

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Du, Zhixue; Dong, Chaoqing; Ren, Jicun

2017-06-01

PTEN (phosphatase and tensin homolog on chromosome 10) is one of the most important tumor-suppressor proteins, which plays a key role in negative regulation of the PI3K/AKT pathway, and governs many cellular processes including growth, proliferation, survival and migration. The dynamics of PTEN proteins in single living cells is as yet unclear owing to a shortage of suitable in vivo approaches. Here, we report a single-molecule method for in vivo study of the dynamics of PTEN proteins in living cells using fluorescence correlation spectroscopy (FCS). First, we established a monoclonal H1299 stable cell line expressing enhanced green fluorescent protein (EGFP) and PTEN (EGFP-PTEN) fusion proteins; we then developed an in vivo FCS method to study the dynamics of EGFP-PTEN both in the nucleus and the cytoplasm. We investigated the diffusion behaviors of EGFP and EGFP-PTEN in solution, nucleus and cytosol, and observed that the motion of PTEN in living cells was restricted compared with EGFP. Finally, we investigated the protein dynamics in living cells under oxidative stress stimulation and a cellular ATP depletion treatment. Under oxidative stress stimulation, the EGFP-PTEN concentration increased in the nucleus, but slightly decreased in the cytoplasm. The diffusion coefficient and alpha value of EGFP-PTEN reduced significantly both in the nucleus and cytoplasm; the significantly decreased alpha parameter indicates a more restricted Brownian diffusion behavior. Under the cellular ATP depletion treatment, the concentration of EGFP-PTEN remained unchanged in the nucleus and decreased significantly in cytosol. The diffusion coefficient of EGFP-PTEN decreased significantly in cytosol, but showed no significant change in the nucleus; the alpha value decreased significantly in both the nucleus and cytoplasm. These results suggest that the concentration and mobility of PTEN in the nucleus and cytoplasm can be regulated by stimulation methods. Our approach provides a unique

Live Cell Characterization of DNA Aggregation Delivered through Lipofection.

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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.

Differentiation of a bipotential glial progenitor cell in a single cell microculture.

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Temple, S; Raff, M C

Although it is known that most cells of the vertebrate central nervous system (CNS) are derived from the neuroepithelial cells of the neural tube, the factors determining whether an individual neuroepithelial cell develops into a particular type of neurone or glial cell remain unknown. A promising model for studying this problem is the bipotential glial progenitor cell in the developing rat optic nerve; this cell differentiates into a particular type of astrocyte (a type-2 astrocyte) if cultured in 10% fetal calf serum (FCS) and into an oligodendrocyte if cultured in serum-free medium. As the oligodendrocyte-type-2 astrocyte (0-2A) progenitor cell can differentiate along either glial pathway in neurone-free cultures, living axons clearly are not required for its differentiation, at least in vitro. However, the studies on 0-2A progenitor cells were carried out in bulk cultures of optic nerve, and so it was possible that other cell-cell interactions were required for differentiation in culture. We show here that 0-2A progenitor cells can differentiate into type-2 astrocytes or oligodendrocytes when grown as isolated cells in microculture, indicating that differentiation along either glial pathway in vitro does not require signals from other CNS cells, apart from the signals provided by components of the culture medium. We also show that single 0-2A progenitor cells can differentiate along either pathway without dividing, supporting our previous studies using 3H-thymidine and suggesting that DNA replication is not required for these cells to choose between the two differentiation programmes.

Compare analysis for the nanotoxicity effects of different amounts of endocytic iron oxide nanoparticles at single cell level.

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Huang, Chen-Yu; Ger, Tzong-Rong; Wei, Zung-Hang; Lai, Mei-Feng

2014-01-01

Developing methods that evaluate the cellular uptake of magnetic nanoparticles (MNPs) and nanotoxicity effects at single-cellular level are needed. In this study, magnetophoresis combining fluorescence based cytotoxicity assay was proposed to assess the viability and the single-cellular MNPs uptake simultaneously. Malignant cells (SKHep-1, HepG2, HeLa) were incubated with 10 nm anionic iron oxide nanoparticles. Prussian blue stain was performed to visualize the distribution of magnetic nanoparticles. MTT and fluorescence based assay analyzed the cytotoxicity effects of the bulk cell population and single cell, respectively. DAPI/PI stained was applied to evaluate death mechanism. The number of intracellular MNPs was found to be strongly correlated with the cell death. Significant differences between cellular MNP uptake in living and dead cells were observed. The method could be useful for future study of the nanotoxicity induced by MNPs.

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

Sets of RNA repeated tags and hybridization-sensitive fluorescent probes for distinct images of RNA in a living cell.

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Takeshi Kubota

Full Text Available BACKGROUND: Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. METHODOLOGY/PRINCIPAL FINDINGS: Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3'-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag-probe pairs. CONCLUSIONS/SIGNIFICANCE: A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.

High-dimensional single-cell cancer biology.

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Irish, Jonathan M; Doxie, Deon B

2014-01-01

Cancer cells are distinguished from each other and from healthy cells by features that drive clonal evolution and therapy resistance. New advances in high-dimensional flow cytometry make it possible to systematically measure mechanisms of tumor initiation, progression, and therapy resistance on millions of cells from human tumors. Here we describe flow cytometry techniques that enable a "single-cell " view of cancer. High-dimensional techniques like mass cytometry enable multiplexed single-cell analysis of cell identity, clinical biomarkers, signaling network phospho-proteins, transcription factors, and functional readouts of proliferation, cell cycle status, and apoptosis. This capability pairs well with a signaling profiles approach that dissects mechanism by systematically perturbing and measuring many nodes in a signaling network. Single-cell approaches enable study of cellular heterogeneity of primary tissues and turn cell subsets into experimental controls or opportunities for new discovery. Rare populations of stem cells or therapy-resistant cancer cells can be identified and compared to other types of cells within the same sample. In the long term, these techniques will enable tracking of minimal residual disease (MRD) and disease progression. By better understanding biological systems that control development and cell-cell interactions in healthy and diseased contexts, we can learn to program cells to become therapeutic agents or target malignant signaling events to specifically kill cancer cells. Single-cell approaches that provide deep insight into cell signaling and fate decisions will be critical to optimizing the next generation of cancer treatments combining targeted approaches and immunotherapy.

Detection and quantification of live, apoptotic, and necrotic human peripheral lymphocytes by single-laser flow cytometry.

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Liegler, T J; Hyun, W; Yen, T S; Stites, D P

1995-05-01

Regulation of peripheral lymphocyte number involves a poorly understood balance between cell renewal and loss. Disrupting this balance leads to a large number of disease states. Methods which allow qualitative and quantitative measurements of cell viability are increasingly valuable to studies directed at revealing the mechanisms underlying apoptotic and necrotic cell death. Here, we have characterized a method using single-laser flow cytometry that differentiates and quantifies the relative number of live, apoptotic, and late-stage apoptotic and necrotic peripheral lymphocytes. Following in vitro gamma irradiation and staining with acridine orange in combination with ethidium bromide, three distinct populations were seen by bivariate analysis of green versus red fluorescence. The identity of each distinct fluorescent population (whether live, apoptotic, or necrotic) was determined by sorting and examination of cellular morphology by electron microscopy. This flow cytometric method is directly compared with the techniques of trypan blue exclusion and DNA fragmentation to quantify cell death following exposure to various doses of in vitro gamma irradiation and postirradiation incubation times. We extend our findings to illustrate the utility of this method beyond analyzing radiation-induced apoptotic peripheral blood mononuclear cells (PBMC); similar fluorescent patterns are shown for radiation- and corticosteroid-treated murine thymocytes, activated human PBMC, and PBMC from human immunodeficiency virus-infected individuals. Our results demonstrate that dual-parameter flow cytometric analysis of acridine orange-ethidium bromide-stained lymphocytes is overall a superior method with increased sensitivity, greater accuracy, and decreased subjectivity in comparison with the other methods tested. By using standard laser and filter settings commonly available to flow cytometric laboratories, this method allows rapid measurement of a large number of cells from a

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

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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

Cellular organization and spectral diversity of GFP-like proteins in live coral cells studied by single and multiphoton imaging and microspectroscopy

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Salih, Anya; Cox, Guy C.; Larkum, Anthony W.

2003-07-01

Tissues of many marine invertebrates of class Anthozoa contain intensely fluorescent or brightly coloured pigments. These pigments belong to a family of photoactive proteins closely related to Green Fluorescent Protein (GFP), and their emissions range from blue to red wavelengths. The great diversity of these pigments has only recently been realised. To investigate the role of these proteins in corals, we have performed an in vivo fluorescent pigment (FP) spectral and cellular distribution analyses in live coral cells using single and multi-photon laser scanning imaging and microspectroscopy. These analyses revealed that even single colour corals contain spectroscopically heterogeneous pigment mixtures, with 2-5 major colour types in the same area of tissue. They were typically arranged in step-wise light emission energy gradients (e.g. blue, green, yellow, red). The successive overlapping emission-excitation spectral profiles of differently coloured FPs suggested that they were suited for sequential energy coupling. Traces of red FPs (emission = 570-660 nm) were present, even in non-red corals. We confirmed that radiative energy transfer could occur between separate granules of blue and green FPs and that energy transfer was inversely proportional to the square of the distance between them. Multi-photon micro-spectrofluorometric analysis gave significantly improved spectral resolution by restricting FP excitation to a single point in the focal plane of the sample. Pigment heterogeneity at small scales within granules suggested that fluorescence resonance energy transfer (FRET) might be occurring, and we confirmed that this was the case. Thus, energy transfer can take place both radiatively and by FRET, probably functioning in photoprotection by dissipation of excessive solar radiation.

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

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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.

Single-cell real-time imaging of transgene expression upon lipofection.

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Fiume, Giuseppe; Di Rienzo, Carmine; Marchetti, Laura; Pozzi, Daniela; Caracciolo, Giulio; Cardarelli, Francesco

2016-05-20

Here we address the process of lipofection by quantifying the expression of a genetically-encoded fluorescent reporter at the single-cell level, and in real-time, by confocal imaging in live cells. The Lipofectamine gold-standard formulation is compared to the alternative promising DC-Chol/DOPE formulation. In both cases, we report that only dividing cells are able to produce a detectable amount of the fluorescent reporter protein. Notably, by measuring fluorescence over time in each pair of daughter cells, we find that Lipofectamine-based transfection statistically yields a remarkably higher degree of "symmetry" in protein expression between daughter cells as compared to DC-Chol/DOPE. A model is envisioned in which the degree of symmetry of protein expression is linked to the number of bioavailable DNA copies within the cell before nuclear breakdown. Reported results open new perspectives for the understanding of the lipofection mechanism and define a new experimental platform for the quantitative comparison of transfection reagents. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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.

Atomic force microscopy stiffness tomography on living Arabidopsis thaliana cells reveals the mechanical properties of surface and deep cell-wall layers during growth.

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Radotić, Ksenija; Roduit, Charles; Simonović, Jasna; Hornitschek, Patricia; Fankhauser, Christian; Mutavdžić, Dragosav; Steinbach, Gabor; Dietler, Giovanni; Kasas, Sandor

2012-08-08

Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

High-frequency microrheology reveals cytoskeleton dynamics in living cells

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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.

Multiphoton-generated localized electron plasma for membrane permeability modification in single cells

Science.gov (United States)

Merritt, T.; Leblanc, M.; McMillan, J.; Westwood, J.; Khodaparast, G. A.

2014-03-01

Successful incorporation of a specific macromolecule into a single cell would be ideal for characterizing trafficking dynamics through plasmodesmata or for studying intracellular localizations. Here, we demonstrate NIR femtosecond laser-mediated infiltration of a membrane impermeable dextran-conjugated dye into living cells of Arabidopsis thaliana seedling stems. Based on the reactions of fluorescing vacuoles of transgenic cells and artificial cell walls comprised of nanocellulose, laser intensity and exposure time were adjusted to avoid deleterious effects. Using these plant-tailored laser parameters, cells were injected with the fluorophores and long-term dye retention was observed, all while preserving vital cell functions. This method is ideal for studies concerning cell-to-cell interactions and potentially paves the way for introducing transgenes to specific cells. This work was supported by NSF award IOS-0843372 to JHW, with additional support from and U.S. Department of Agriculture Hatch Project no. 135997, and by the Institute of Critical Technology and Applied Sciences (ICTAS) at Virginia Tech.

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.

Single-cell regulome data analysis by SCRAT.

Science.gov (United States)

Ji, Zhicheng; Zhou, Weiqiang; Ji, Hongkai

2017-09-15

Emerging single-cell technologies (e.g. single-cell ATAC-seq, DNase-seq or ChIP-seq) have made it possible to assay regulome of individual cells. Single-cell regulome data are highly sparse and discrete. Analyzing such data is challenging. User-friendly software tools are still lacking. We present SCRAT, a Single-Cell Regulome Analysis Toolbox with a graphical user interface, for studying cell heterogeneity using single-cell regulome data. SCRAT can be used to conveniently summarize regulatory activities according to different features (e.g. gene sets, transcription factor binding motif sites, etc.). Using these features, users can identify cell subpopulations in a heterogeneous biological sample, infer cell identities of each subpopulation, and discover distinguishing features such as gene sets and transcription factors that show different activities among subpopulations. SCRAT is freely available at https://zhiji.shinyapps.io/scrat as an online web service and at https://github.com/zji90/SCRAT as an R package. hji@jhu.edu. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell.

Science.gov (United States)

Nagano, Takashi; Lubling, Yaniv; Yaffe, Eitan; Wingett, Steven W; Dean, Wendy; Tanay, Amos; Fraser, Peter

2015-12-01

Hi-C is a powerful method that provides pairwise information on genomic regions in spatial proximity in the nucleus. Hi-C requires millions of cells as input and, as genome organization varies from cell to cell, a limitation of Hi-C is that it only provides a population average of genome conformations. We developed single-cell Hi-C to create snapshots of thousands of chromatin interactions that occur simultaneously in a single cell. To adapt Hi-C to single-cell analysis, we modified the protocol to include in-nucleus ligation. This enables the isolation of single nuclei carrying Hi-C-ligated DNA into separate tubes, followed by reversal of cross-links, capture of biotinylated ligation junctions on streptavidin-coated magnetic beads and PCR amplification of single-cell Hi-C libraries. The entire laboratory protocol can be carried out in 1 week, and although we have demonstrated its use in mouse T helper (TH1) cells, it should be applicable to any cell type or species for which standard Hi-C has been successful. We also developed an analysis pipeline to filter noise and assess the quality of data sets in a few hours. Although the interactome maps produced by single-cell Hi-C are sparse, the data provide useful information to understand cellular variability in nuclear genome organization and chromosome structure. Standard wet and dry laboratory skills in molecular biology and computational analysis are required.

Metabolomic Fingerprints of Individual Algal Cells Using the Single-Probe Mass Spectrometry Technique

Directory of Open Access Journals (Sweden)

Mei Sun

2018-04-01

Full Text Available Traditional approaches for the assessment of physiological responses of microbes in the environment rely on bulk filtration techniques that obscure differences among populations as well as among individual cells. Here, were report on the development on a novel micro-scale sampling device, referred to as the “Single-probe,” which allows direct extraction of metabolites from living, individual phytoplankton cells for mass spectrometry (MS analysis. The Single-probe is composed of dual-bore quartz tubing which is pulled using a laser pipette puller and fused to a silica capillary and a nano-ESI. For this study, we applied Single-probe MS technology to the marine dinoflagellate Scrippsiella trochoidea, assaying cells grown under different illumination levels and under nitrogen (N limiting conditions as a proof of concept for the technology. In both experiments, significant differences in the cellular metabolome of individual cells could readily be identified, though the vast majority of detected metabolites could not be assigned to KEGG pathways. Using the same approach, significant changes in cellular lipid complements were observed, with individual lipids being both up- and down-regulated under light vs. dark conditions. Conversely, lipid content increased across the board under N limitation, consistent with an adjustment of Redfield stoichiometry to reflect higher C:N and C:P ratios. Overall, these data suggest that the Single-probe MS technique has the potential to allow for near in situ metabolomic analysis of individual phytoplankton cells, opening the door to targeted analyses that minimize cell manipulation and sampling artifacts, while preserving metabolic variability at the cellular level.

The Caenorhabditis elegans Q neuroblasts: A powerful system to study cell migration at single-cell resolution in vivo.

Science.gov (United States)

Rella, Lorenzo; Fernandes Póvoa, Euclides E; Korswagen, Hendrik C

2016-04-01

During development, cell migration plays a central role in the formation of tissues and organs. Understanding the molecular mechanisms that drive and control these migrations is a key challenge in developmental biology that will provide important insights into disease processes, including cancer cell metastasis. In this article, we discuss the Caenorhabditis elegans Q neuroblasts and their descendants as a tool to study cell migration at single-cell resolution in vivo. The highly stereotypical migration of these cells provides a powerful system to study the dynamic cytoskeletal processes that drive migration as well as the evolutionarily conserved signaling pathways (including different Wnt signaling cascades) that guide the cells along their specific trajectories. Here, we provide an overview of what is currently known about Q neuroblast migration and highlight the live-cell imaging, genome editing, and quantitative gene expression techniques that have been developed to study this process. © 2016 Wiley Periodicals, Inc.

Long-Term Live Cell Imaging Reveals New Roles For Salmonella Effector Proteins SseG and SteA

Science.gov (United States)

McQuate, Sarah E.; Young, Alexandra M.; Silva-Herzog, Eugenia; Bunker, Eric; Hernandez, Mateo; de Chaumont, Fabrice; Liu, Xuedong; Detweiler, Corrella S.; Palmer, Amy E.

2016-01-01

Summary Salmonella Typhimurium is an intracellular bacterial pathogen that infects both epithelial cells and macrophages. Salmonella effector proteins, which are translocated into the host cell and manipulate host cell components, control the ability to replicate and/or survive in host cells. Due to the complexity and heterogeneity of Salmonella infections, there is growing recognition of the need for single cell and live-cell imaging approaches to identify and characterize the diversity of cellular phenotypes and how they evolve over time. Here we establish a pipeline for long-term (16 hours) live-cell imaging of infected cells and subsequent image analysis methods. We apply this pipeline to track bacterial replication within the Salmonella-containing vacuole in epithelial cells, quantify vacuolar replication versus survival in macrophages, and investigate the role of individual effector proteins in mediating these parameters. This approach revealed that dispersed bacteria can coalesce at later stages of infection, that the effector protein SseG influences the propensity for cytosolic hyperreplication in epithelial cells, and that while SteA only has a subtle effect on vacuolar replication in epithelial cells, it has a profound impact on infection parameters in immunocompetent macrophages, suggesting differential roles for effector proteins in different infection models. PMID:27376507

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.

Single-cell technologies in environmental omics

KAUST Repository

Kodzius, Rimantas

2015-10-22

Environmental studies are primarily done by culturing isolated microorganisms or by amplifying and sequencing conserved genes. Difficulties understanding the complexity of large numbers of various microorganisms in an environment led to the development of techniques to enrich specific microorganisms for upstream analysis, ultimately leading to single-cell isolation and analyses. We discuss the significance of single-cell technologies in omics studies with focus on metagenomics and metatranscriptomics. We propose that by reducing sample heterogeneity using single-cell genomics, metaomic studies can be simplified.

Single-cell technologies in environmental omics

KAUST Repository

Kodzius, Rimantas; Gojobori, Takashi

2015-01-01

Environmental studies are primarily done by culturing isolated microorganisms or by amplifying and sequencing conserved genes. Difficulties understanding the complexity of large numbers of various microorganisms in an environment led to the development of techniques to enrich specific microorganisms for upstream analysis, ultimately leading to single-cell isolation and analyses. We discuss the significance of single-cell technologies in omics studies with focus on metagenomics and metatranscriptomics. We propose that by reducing sample heterogeneity using single-cell genomics, metaomic studies can be simplified.

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

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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

Single-cell measurement of red blood cell oxygen affinity

OpenAIRE

Caprio, Di; Stokes, Chris; Higgins, John M.; Schonbrun, Ethan

2015-01-01

Oxygen is transported throughout the body by hemoglobin in red blood cells. While the oxygen affinity of blood is well understood and is routinely assessed in patients by pulse oximetry, variability at the single-cell level has not been previously measured. In contrast, single-cell measurements of red blood cell volume and hemoglobin concentration are taken millions of times per day by clinical hematology analyzers and are important factors in determining the health of the hematologic system....

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

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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

Single living predicts a higher mortality in both women and men with chronic heart failure.

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Mard, Shan; Nielsen, Finn Erland

2016-09-01

We examined the impact of single living on all-cause mortality in patients with chronic heart failure and determined if this association was modified by gender. This historical cohort study included 637 patients who were admitted to the Department of Cardiology, Herlev Hospital, Denmark, between 1 July 2005 and 30 June 2007. Baseline clinical data were obtained from patient records. Data on survival rates were obtained from the Danish Civil Registration System. Cox proportional hazard analysis was used to compute the hazard ratio (HR) of all-cause mortality, controlling for confounding factors. The median follow-up time was 2.8 years. A total of 323 (50.7%) patients died during the follow-up period. After adjustment for confounding factors, risk of death was associated with being single (HR = 1.53 (95% confidence interval: 1.19-1.96)). In a gender-stratified analysis, the risk of death did not differ among single-living women and men. Single living is a prognostic determinant of all-cause mortality in men and women with chronic heart failure. none. not relevant.

Vibrational imaging of glucose uptake activity in live cells and tissues by stimulated Raman scattering microscopy (Conference Presentation)

Science.gov (United States)

Hu, Fanghao; Chen, Zhixing; Zhang, Luyuan; Shen, Yihui; Wei, Lu; Min, Wei

2016-03-01

Glucose is consumed as an energy source by virtually all living organisms, from bacteria to humans. Its uptake activity closely reflects the cellular metabolic status in various pathophysiological transformations, such as diabetes and cancer. Extensive efforts such as positron emission tomography, magnetic resonance imaging and fluorescence microscopy have been made to specifically image glucose uptake activity but all with technical limitations. Here, we report a new platform to visualize glucose uptake activity in live cells and tissues with subcellular resolution and minimal perturbation. A novel glucose analogue with a small alkyne tag (carbon-carbon triple bond) is developed to mimic natural glucose for cellular uptake, which can be imaged with high sensitivity and specificity by targeting the strong and characteristic alkyne vibration on stimulated Raman scattering (SRS) microscope to generate a quantitative three dimensional concentration map. Cancer cells with differing metabolic characteristics can be distinguished. Heterogeneous uptake patterns are observed in tumor xenograft tissues, neuronal culture and mouse brain tissues with clear cell-cell variations. Therefore, by offering the distinct advantage of optical resolution but without the undesirable influence of bulky fluorophores, our method of coupling SRS with alkyne labeled glucose will be an attractive tool to study energy demands of living systems at the single cell level.

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

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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

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.

Single cell enzyme diagnosis on the chip

DEFF Research Database (Denmark)

Jensen, Sissel Juul; Harmsen, Charlotte; Nielsen, Mette Juul

2013-01-01

Conventional diagnosis based on ensemble measurements often overlooks the variation among cells. Here, we present a droplet-microfluidics based platform to investigate single cell activities. Adopting a previously developed isothermal rolling circle amplification-based assay, we demonstrate...... detection of enzymatic activities down to the single cell level with small quantities of biological samples, which outcompetes existing techniques. Such a system, capable of resolving single cell activities, will ultimately have clinical applications in diagnosis, prediction of drug response and treatment...

Circumventing photodamage in live-cell microscopy

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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

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.

Near-Infrared Light Activation of Proteins Inside Living Cells Enabled by Carbon Nanotube-Mediated Intracellular Delivery.

Science.gov (United States)

Li, He; Fan, Xinqi; Chen, Xing

2016-02-01

Light-responsive proteins have been delivered into the cells for controlling intracellular events with high spatial and temporal resolution. However, the choice of wavelength is limited to the UV and visible range; activation of proteins inside the cells using near-infrared (NIR) light, which has better tissue penetration and biocompatibility, remains elusive. Here, we report the development of a single-walled carbon nanotube (SWCNT)-based bifunctional system that enables protein intracellular delivery, followed by NIR activation of the delivered proteins inside the cells. Proteins of interest are conjugated onto SWCNTs via a streptavidin-desthiobiotin (SA-DTB) linkage, where the protein activity is blocked. SWCNTs serve as both a nanocarrier for carrying proteins into the cells and subsequently a NIR sensitizer to photothermally cleave the linkage and release the proteins. The released proteins become active and exert their functions inside the cells. We demonstrated this strategy by intracellular delivery and NIR-triggered nuclear translocation of enhanced green fluorescent protein, and by intracellular delivery and NIR-activation of a therapeutic protein, saporin, in living cells. Furthermore, we showed that proteins conjugated onto SWCNTs via the SA-DTB linkage could be delivered to the tumors, and optically released and activated by using NIR light in living mice.

Platforms for Single-Cell Collection and Analysis

Directory of Open Access Journals (Sweden)

Lukas Valihrach

2018-03-01

Full Text Available Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS. In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.

Platforms for Single-Cell Collection and Analysis.

Science.gov (United States)

Valihrach, Lukas; Androvic, Peter; Kubista, Mikael

2018-03-11

Single-cell analysis has become an established method to study cell heterogeneity and for rare cell characterization. Despite the high cost and technical constraints, applications are increasing every year in all fields of biology. Following the trend, there is a tremendous development of tools for single-cell analysis, especially in the RNA sequencing field. Every improvement increases sensitivity and throughput. Collecting a large amount of data also stimulates the development of new approaches for bioinformatic analysis and interpretation. However, the essential requirement for any analysis is the collection of single cells of high quality. The single-cell isolation must be fast, effective, and gentle to maintain the native expression profiles. Classical methods for single-cell isolation are micromanipulation, microdissection, and fluorescence-activated cell sorting (FACS). In the last decade several new and highly efficient approaches have been developed, which not just supplement but may fully replace the traditional ones. These new techniques are based on microfluidic chips, droplets, micro-well plates, and automatic collection of cells using capillaries, magnets, an electric field, or a punching probe. In this review we summarize the current methods and developments in this field. We discuss the advantages of the different commercially available platforms and their applicability, and also provide remarks on future developments.

A Comparison Study of Single-Parent Families Living on Remote, Rural Islands and in Urban Settings in Japan.

Science.gov (United States)

Hiratani, Yuko; Hohashi, Naohiro

2016-06-01

Nursing interventions that aim to enhance the family environment are necessary to help single-parent families with children to improve family functioning. The cultural and social factors that are unique to Japan's remote islands should be considered to assess the influence of this unique setting on family functioning. The objectives of this study were to evaluate the family functioning of child-rearing single-parent families living in different environments and to investigate the association between family demographics and family functioning. A self-administered questionnaire, the Japanese version of the Survey of Family Environment, was used to evaluate the sufficiency of family functioning. The participants were families with children enrolled in nurseries and kindergartens who were either living in remote, rural islands or in an urban city on the mainland in Japan. Family functioning was significantly higher for single-parent families living on the islands than for those living in the city in terms of media use, participation in community activities, and the collaboration of family members in child-rearing. Family functioning of single-parent families correlated significantly with household income, the parent's gender, family members' health, and family life cycle. Single-parent families living on Japanese offshore islands maintained family functioning through mutual support and the effective use of information technology. Nevertheless, single-parent families require additional support to improve their healthcare and financial situations.

Single cell analysis of normal and leukemic hematopoiesis.

Science.gov (United States)

Povinelli, Benjamin J; Rodriguez-Meira, Alba; Mead, Adam J

2018-02-01

The hematopoietic system is well established as a paradigm for the study of cellular hierarchies, their disruption in disease and therapeutic use in regenerative medicine. Traditional approaches to study hematopoiesis involve purification of cell populations based on a small number of surface markers. However, such population-based analysis obscures underlying heterogeneity contained within any phenotypically defined cell population. This heterogeneity can only be resolved through single cell analysis. Recent advances in single cell techniques allow analysis of the genome, transcriptome, epigenome and proteome in single cells at an unprecedented scale. The application of these new single cell methods to investigate the hematopoietic system has led to paradigm shifts in our understanding of cellular heterogeneity in hematopoiesis and how this is disrupted in disease. In this review, we summarize how single cell techniques have been applied to the analysis of hematopoietic stem/progenitor cells in normal and malignant hematopoiesis, with a particular focus on recent advances in single-cell genomics, including how these might be utilized for clinical application. Copyright © 2017. Published by Elsevier Ltd.

Electrical characterization of single cells using polysilicon wire ion sensor in an isolation window.

Science.gov (United States)

Wu, You-Lin; Hsu, Po-Yen; Hsu, Chung-Ping; Wang, Chih-Cheng; Lee, Li-Wen; Lin, Jing-Jenn

2011-10-01

A polysilicon wire (PSW) sensor can detect the H(+) ion density (pH value) of the medium coated on its surface, and different cells produce different extracellular acidification and hence different H(+) ion densities. Based on this, we used a PSW sensor in combination with a mold-cast polydimethylsiloxane (PDMS) isolation window to detect the adhesion, apoptosis and extracellular acidification of single normal cells and single cancer cells. Single living human normal cells WI38, MRC5, and BEAS-2B as well as non-small-cell lung cancer (NSCLC) cells A549, H1299, and CH27 were cultivated separately inside the isolation window. The current flowing through the PSW channel was measured. From the PSW channel current change as a function of time, we determined the cell adhesion time by observing the time required for the current change to saturate, since a stable extracellular ion density was established after the cells were completely adhered to the PSW surface. The apoptosis of cells can also be determined when the channel current change drops to zero. We found that all the NSCLC cells had a higher channel current change and hence a lower pH value than the normal cells anytime after they were seeded. The corresponding average pH values were 5.86 for A549, 6.00 for H1299, 6.20 for CH27, 6.90 for BEAS-2B, 6.96for MRC5, and 7.02 for WI38, respectively, after the cells were completely adhered to the PSW surface. Our results show that NSCLC cells have a stronger cell-substrate adhesion and a higher extracellular acidification rate than normal cells.

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

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

Directory of Open Access Journals (Sweden)

Piotrowska-Nitsche Karolina

2012-05-01

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

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%).

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.

Application of single-cell technology in cancer research.

Science.gov (United States)

Liang, Shao-Bo; Fu, Li-Wu

2017-07-01

In this review, we have outlined the application of single-cell technology in cancer research. Single-cell technology has made encouraging progress in recent years and now provides the means to detect rare cancer cells such as circulating tumor cells and cancer stem cells. We reveal how this technology has advanced the analysis of intratumor heterogeneity and tumor epigenetics, and guided individualized treatment strategies. The future prospects now are to bring single-cell technology into the clinical arena. We believe that the clinical application of single-cell technology will be beneficial in cancer diagnostics and treatment, and ultimately improve survival in cancer patients. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

A high content, high throughput cellular thermal stability assay for measuring drug-target engagement in living cells.

Science.gov (United States)

Massey, Andrew J

2018-01-01

Determining and understanding drug target engagement is critical for drug discovery. This can be challenging within living cells as selective readouts are often unavailable. Here we describe a novel method for measuring target engagement in living cells based on the principle of altered protein thermal stabilization / destabilization in response to ligand binding. This assay (HCIF-CETSA) utilizes high content, high throughput single cell immunofluorescent detection to determine target protein levels following heating of adherent cells in a 96 well plate format. We have used target engagement of Chk1 by potent small molecule inhibitors to validate the assay. Target engagement measured by this method was subsequently compared to target engagement measured by two alternative methods (autophosphorylation and CETSA). The HCIF-CETSA method appeared robust and a good correlation in target engagement measured by this method and CETSA for the selective Chk1 inhibitor V158411 was observed. However, these EC50 values were 23- and 12-fold greater than the autophosphorylation IC50. The described method is therefore a valuable advance in the CETSA method allowing the high throughput determination of target engagement in adherent cells.

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.

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

DNA double strand breaks and Hsp70 expression in proton irradiated living cells

International Nuclear Information System (INIS)

Fiedler, Anja; Reinert, Tilo; Tanner, Judith; Butz, Tilman

2007-01-01

DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone γH2AX. Our concern was to test the feasibility of γH2AX staining for a direct visualization of single proton hits. If single protons produce detectable foci, DNA DSBs could be used as 'biological track detectors' for protons. Ionising radiation can also damage proteins indirectly by inducing free radicals. Heat shock proteins (Hsp) help to refold or even degrade the damaged proteins. The level of the most famous heat shock protein Hsp70 is increased by ionising radiation. We investigated the expression of γH2AX and Hsp70 after cross and line patterned irradiation with counted numbers of 2.25 MeV protons on primary human skin fibroblasts. The proton induced DSBs appear more delocalised than it was expected by the ion hit accuracy. Cooling the cells before the irradiation reduces the delocalisation of DNA DSBs, which is probably caused by the reduced diffusion of DNA damaging agents. Proton irradiation seems to provoke protein damages mainly in the cytoplasm indicated by cytoplasmic Hsp70 aggregates. On the contrary, in control heat shocked cells the Hsp70 was predominantly localized in the cell nucleus. However, the irradiated area could not be recognized, all cells on the Si 3 N 4 window showed a homogenous Hsp70 expression pattern

DNA double strand breaks and Hsp70 expression in proton irradiated living cells

Energy Technology Data Exchange (ETDEWEB)

Fiedler, Anja [Institute for Experimental Physics II, University of Leipzig (Germany) and Faculty of Biology, Pharmacy and Psychology, University of Leipzig (Germany)]. E-mail: afiedler@uni-leipzig.de; Reinert, Tilo [Institute for Experimental Physics II, University of Leipzig (Germany); Tanner, Judith [Clinic and Polyclinic for Radiation Oncology, University of Halle-Wittenberg (Germany); Butz, Tilman [Institute for Experimental Physics II, University of Leipzig (Germany)

2007-07-15

DNA double strand breaks (DSBs) in living cells can be directly provoked by ionising radiation. DSBs can be visualized by immunostaining the phosphorylated histone {gamma}H2AX. Our concern was to test the feasibility of {gamma}H2AX staining for a direct visualization of single proton hits. If single protons produce detectable foci, DNA DSBs could be used as 'biological track detectors' for protons. Ionising radiation can also damage proteins indirectly by inducing free radicals. Heat shock proteins (Hsp) help to refold or even degrade the damaged proteins. The level of the most famous heat shock protein Hsp70 is increased by ionising radiation. We investigated the expression of {gamma}H2AX and Hsp70 after cross and line patterned irradiation with counted numbers of 2.25 MeV protons on primary human skin fibroblasts. The proton induced DSBs appear more delocalised than it was expected by the ion hit accuracy. Cooling the cells before the irradiation reduces the delocalisation of DNA DSBs, which is probably caused by the reduced diffusion of DNA damaging agents. Proton irradiation seems to provoke protein damages mainly in the cytoplasm indicated by cytoplasmic Hsp70 aggregates. On the contrary, in control heat shocked cells the Hsp70 was predominantly localized in the cell nucleus. However, the irradiated area could not be recognized, all cells on the Si{sub 3}N{sub 4} window showed a homogenous Hsp70 expression pattern.

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.

Live-cell microscopy reveals small molecule inhibitor effects on MAPK pathway dynamics.

Directory of Open Access Journals (Sweden)

Daniel J Anderson

Full Text Available Oncogenic mutations in the mitogen activated protein kinase (MAPK pathway are prevalent in human tumors, making this pathway a target of drug development efforts. Recently, ATP-competitive Raf inhibitors were shown to cause MAPK pathway activation via Raf kinase priming in wild-type BRaf cells and tumors, highlighting the need for a thorough understanding of signaling in the context of small molecule kinase inhibitors. Here, we present critical improvements in cell-line engineering and image analysis coupled with automated image acquisition that allow for the simultaneous identification of cellular localization of multiple MAPK pathway components (KRas, CRaf, Mek1 and Erk2. We use these assays in a systematic study of the effect of small molecule inhibitors across the MAPK cascade either as single agents or in combination. Both Raf inhibitor priming as well as the release from negative feedback induced by Mek and Erk inhibitors cause translocation of CRaf to the plasma membrane via mechanisms that are additive in pathway activation. Analysis of Erk activation and sub-cellular localization upon inhibitor treatments reveals differential inhibition and activation with the Raf inhibitors AZD628 and GDC0879 respectively. Since both single agent and combination studies of Raf and Mek inhibitors are currently in the clinic, our assays provide valuable insight into their effects on MAPK signaling in live cells.

[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.

Automated Single Cell Data Decontamination Pipeline

Energy Technology Data Exchange (ETDEWEB)

Tennessen, Kristin [Lawrence Berkeley National Lab. (LBNL), Walnut Creek, CA (United States). Dept. of Energy Joint Genome Inst.; Pati, Amrita [Lawrence Berkeley National Lab. (LBNL), Walnut Creek, CA (United States). Dept. of Energy Joint Genome Inst.

2014-03-21

Recent technological advancements in single-cell genomics have encouraged the classification and functional assessment of microorganisms from a wide span of the biospheres phylogeny.1,2 Environmental processes of interest to the DOE, such as bioremediation and carbon cycling, can be elucidated through the genomic lens of these unculturable microbes. However, contamination can occur at various stages of the single-cell sequencing process. Contaminated data can lead to wasted time and effort on meaningless analyses, inaccurate or erroneous conclusions, and pollution of public databases. A fully automated decontamination tool is necessary to prevent these instances and increase the throughput of the single-cell sequencing process

Immobilisation of barley aleurone layers enables parallelisation of assays and analysis of transient gene expression in single cells

DEFF Research Database (Denmark)

Zor, Kinga; Mark, Christina; Heiskanen, Arto

2017-01-01

at a single time point. By immobilising barley aleurone layer tissue on polydimethylsiloxane pillars in the lid of a multiwell plate, continuous monitoring of living tissue is enabled using multiple non-destructive assays in parallel. Cell viability and menadione reducing capacity were monitored in the same...

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.

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.

Single cell metabolomics

NARCIS (Netherlands)

Heinemann, Matthias; Zenobi, Renato

Recent discoveries suggest that cells of a clonal population often display multiple metabolic phenotypes at the same time. Motivated by the success of mass spectrometry (MS) in the investigation of population-level metabolomics, the analytical community has initiated efforts towards MS-based single

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.

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

Energy Technology Data Exchange (ETDEWEB)

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

2008-12-18

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

Single-cell intracellular nano-pH probes†

Science.gov (United States)

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2016-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution. PMID:27708772

Single-cell intracellular nano-pH probes.

Science.gov (United States)

Özel, Rıfat Emrah; Lohith, Akshar; Mak, Wai Han; Pourmand, Nader

2015-01-01

Within a large clonal population, such as cancerous tumor entities, cells are not identical, and the differences between intracellular pH levels of individual cells may be important indicators of heterogeneity that could be relevant in clinical practice, especially in personalized medicine. Therefore, the detection of the intracellular pH at the single-cell level is of great importance to identify and study outlier cells. However, quantitative and real-time measurements of the intracellular pH of individual cells within a cell population is challenging with existing technologies, and there is a need to engineer new methodologies. In this paper, we discuss the use of nanopipette technology to overcome the limitations of intracellular pH measurements at the single-cell level. We have developed a nano-pH probe through physisorption of chitosan onto hydroxylated quartz nanopipettes with extremely small pore sizes (~100 nm). The dynamic pH range of the nano-pH probe was from 2.6 to 10.7 with a sensitivity of 0.09 units. We have performed single-cell intracellular pH measurements using non-cancerous and cancerous cell lines, including human fibroblasts, HeLa, MDA-MB-231 and MCF-7, with the pH nanoprobe. We have further demonstrated the real-time continuous single-cell pH measurement capability of the sensor, showing the cellular pH response to pharmaceutical manipulations. These findings suggest that the chitosan-functionalized nanopore is a powerful nano-tool for pH sensing at the single-cell level with high temporal and spatial resolution.

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.

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

mRNA-Seq of single prostate cancer circulating tumor cells reveals recapitulation of gene expression and pathways found in prostate cancer.

Science.gov (United States)

Cann, Gordon M; Gulzar, Zulfiqar G; Cooper, Samantha; Li, Robin; Luo, Shujun; Tat, Mai; Stuart, Sarah; Schroth, Gary; Srinivas, Sandhya; Ronaghi, Mostafa; Brooks, James D; Talasaz, Amirali H

2012-01-01

Circulating tumor cells (CTC) mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

Science.gov (United States)

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

Imaging Intracellular pH in Live Cells with a Genetically-Encoded Red Fluorescent Protein Sensor

OpenAIRE

Tantama, Mathew; Hung, Yin Pun; Yellen, Gary

2011-01-01

Intracellular pH affects protein structure and function, and proton gradients underlie the function of organelles such as lysosomes and mitochondria. We engineered a genetically-encoded pH sensor by mutagenesis of the red fluorescent protein mKeima, providing a new tool to image intracellular pH in live cells. This sensor, named pHRed, is the first ratiometric, single-protein red fluorescent sensor of pH. Fluorescence emission of pHRed peaks at 610 nm while exhibiting dual excitation peaks at...

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.

Simple and fast spectral domain algorithm for quantitative phase imaging of living cells with digital holographic microscopy

Science.gov (United States)

Min, Junwei; Yao, Baoli; Ketelhut, Steffi; Kemper, Björn

2017-02-01

The modular combination of optical microscopes with digital holographic microscopy (DHM) has been proven to be a powerful tool for quantitative live cell imaging. The introduction of condenser and different microscope objectives (MO) simplifies the usage of the technique and makes it easier to measure different kinds of specimens with different magnifications. However, the high flexibility of illumination and imaging also causes variable phase aberrations that need to be eliminated for high resolution quantitative phase imaging. The existent phase aberrations compensation methods either require add additional elements into the reference arm or need specimen free reference areas or separate reference holograms to build up suitable digital phase masks. These inherent requirements make them unpractical for usage with highly variable illumination and imaging systems and prevent on-line monitoring of living cells. In this paper, we present a simple numerical method for phase aberration compensation based on the analysis of holograms in spatial frequency domain with capabilities for on-line quantitative phase imaging. From a single shot off-axis hologram, the whole phase aberration can be eliminated automatically without numerical fitting or pre-knowledge of the setup. The capabilities and robustness for quantitative phase imaging of living cancer cells are demonstrated.

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

Single-cell entropy for accurate estimation of differentiation potency from a cell's transcriptome

Science.gov (United States)

Teschendorff, Andrew E.; Enver, Tariq

2017-01-01

The ability to quantify differentiation potential of single cells is a task of critical importance. Here we demonstrate, using over 7,000 single-cell RNA-Seq profiles, that differentiation potency of a single cell can be approximated by computing the signalling promiscuity, or entropy, of a cell's transcriptome in the context of an interaction network, without the need for feature selection. We show that signalling entropy provides a more accurate and robust potency estimate than other entropy-based measures, driven in part by a subtle positive correlation between the transcriptome and connectome. Signalling entropy identifies known cell subpopulations of varying potency and drug resistant cancer stem-cell phenotypes, including those derived from circulating tumour cells. It further reveals that expression heterogeneity within single-cell populations is regulated. In summary, signalling entropy allows in silico estimation of the differentiation potency and plasticity of single cells and bulk samples, providing a means to identify normal and cancer stem-cell phenotypes. PMID:28569836

Technical aspects and recommendations for single-cell qPCR.

Science.gov (United States)

Ståhlberg, Anders; Kubista, Mikael

2018-02-01

Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories. Copyright © 2017 Elsevier Ltd. All rights reserved.

Photoacoustic bio-quantification of graphene based nanomaterials at a single cell level (Conference Presentation)

Science.gov (United States)

Nedosekin, Dmitry A.; Nolan, Jacqueline; Biris, Alexandru S.; Zharov, Vladimir P.

2017-03-01

Arkansas Nanomedicine Center at the University of Arkansas for Medical Sciences in collaboration with other Arkansas Universities and the FDA-based National Center of Toxicological Research in Jefferson, AR is developing novel techniques for rapid quantification of graphene-based nanomaterials (GBNs) in various biological samples. All-carbon GBNs have wide range of potential applications in industry, agriculture, food processing and medicine; however, quantification of GBNs is difficult in carbon reach biological tissues. The accurate quantification of GBNs is essential for research on material toxicity and the development of GBNs-based drug delivery platforms. We have developed microscopy and cytometry platforms for detection and quantification of GBNs in single cells, tissue and blood samples using photoacoustic contrast of GBNs. We demonstrated PA quantification of individual graphene uptake by single cells. High-resolution PA microscopy provided mapping of GBN distribution within live cells to establish correlation with intracellular toxic phenomena using apoptotic and necrotic assays. This new methodology and corresponding technical platform provide the insight on possible toxicological risks of GBNs at singe cells levels. In addition, in vivo PA image flow cytometry demonstrated the capability to monitor of GBNs pharmacokinetics in mouse model and to map the resulting biodistribution of GBNs in mouse tissues. The integrated PA platform provided an unprecedented sensitivity toward GBNs and allowed to enhance conventional toxicology research by providing a direct correlation between uptake of GBNs at a single cell level and cell viability status.

Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches

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Wu, Jincheng; Tzanakakis, Emmanuel S.

2014-01-01

Isogenic stem cell populations display cell-to-cell variations in a multitude of attributes including gene or protein expression, epigenetic state, morphology, proliferation and proclivity for differentiation. The origins of the observed heterogeneity and its roles in the maintenance of pluripotency and the lineage specification of stem cells remain unclear. Addressing pertinent questions will require the employment of single-cell analysis methods as traditional cell biochemical and biomolecular assays yield mostly population-average data. In addition to time-lapse microscopy and flow cytometry, recent advances in single-cell genomic, transcriptomic and proteomic profiling are reviewed. The application of multiple displacement amplification, next generation sequencing, mass cytometry and spectrometry to stem cell systems is expected to provide a wealth of information affording unprecedented levels of multiparametric characterization of cell ensembles under defined conditions promoting pluripotency or commitment. Establishing connections between single-cell analysis information and the observed phenotypes will also require suitable mathematical models. Stem cell self-renewal and differentiation are orchestrated by the coordinated regulation of subcellular, intercellular and niche-wide processes spanning multiple time scales. Here, we discuss different modeling approaches and challenges arising from their application to stem cell populations. Integrating single-cell analysis with computational methods will fill gaps in our knowledge about the functions of heterogeneity in stem cell physiology. This combination will also aid the rational design of efficient differentiation and reprogramming strategies as well as bioprocesses for the production of clinically valuable stem cell derivatives. PMID:24035899

Identification of innate lymphoid cells in single-cell RNA-Seq data.

Science.gov (United States)

Suffiotti, Madeleine; Carmona, Santiago J; Jandus, Camilla; Gfeller, David

2017-07-01

Innate lymphoid cells (ILCs) consist of natural killer (NK) cells and non-cytotoxic ILCs that are broadly classified into ILC1, ILC2, and ILC3 subtypes. These cells recently emerged as important early effectors of innate immunity for their roles in tissue homeostasis and inflammation. Over the last few years, ILCs have been extensively studied in mouse and human at the functional and molecular level, including gene expression profiling. However, sorting ILCs with flow cytometry for gene expression analysis is a delicate and time-consuming process. Here we propose and validate a novel framework for studying ILCs at the transcriptomic level using single-cell RNA-Seq data. Our approach combines unsupervised clustering and a new cell type classifier trained on mouse ILC gene expression data. We show that this approach can accurately identify different ILCs, especially ILC2 cells, in human lymphocyte single-cell RNA-Seq data. Our new model relies only on genes conserved across vertebrates, thereby making it in principle applicable in any vertebrate species. Considering the rapid increase in throughput of single-cell RNA-Seq technology, our work provides a computational framework for studying ILC2 cells in single-cell transcriptomic data and may help exploring their conservation in distant vertebrate species.

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.

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

Characterisation of a pre-cell hit detector to be used in single cell irradiation experiments at the Lund Nuclear Microprobe

International Nuclear Information System (INIS)

Nilsson, Charlotta; Pallon, Jan; Thungstroem, Goeran; Marrero, Natalia Arteaga; Elfman, Mikael; Kristiansson, Per; Nilsson, Christer; Wegden, Marie

2008-01-01

This paper describes the characterisation of an ultra-thin silicon semiconductor ΔE detector to be used as a pre-cell ion hit detector in single ion experiments on individual, living cells. The characteristics of interest for this specific application are the hit detection efficiency, which has to be close to 100% to enable bombardment with either a single ion or a counted number of ions, the beam spreading, which should be as small as possible to maintain the targeting accuracy, and the vacuum tightness, since the detector is intended, if possible, to be used simultaneously as vacuum window. The hit detection efficiency was shown to be above 99% when detecting alpha particles or 2 MeV protons, the increase in beam size was about 1 μm and the vacuum tightness was comparable to that of the Si 3 N 4 wafer which is normally used as vacuum window, thus the ΔE detector fulfils the main criteria to function properly as a single ion hit detector.

Microchip screening platform for single cell assessment of NK cell cytotoxicity

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Karolin eGuldevall

2016-04-01

Full Text Available Here we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32 400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75% were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3 target cells within the 12 hours long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g. in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

Science.gov (United States)

Guldevall, Karolin; Brandt, Ludwig; Forslund, Elin; Olofsson, Karl; Frisk, Thomas W.; Olofsson, Per E.; Gustafsson, Karin; Manneberg, Otto; Vanherberghen, Bruno; Brismar, Hjalmar; Kärre, Klas; Uhlin, Michael; Önfelt, Björn

2016-01-01

Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy. PMID:27092139

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)

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.

Single-organelle tracking by two-photon conversion

Science.gov (United States)

Watanabe, Wataru; Shimada, Tomoko; Matsunaga, Sachihiro; Kurihara, Daisuke; Fukui, Kiichi; Shin-Ichi Arimura, Shin-Ichi; Tsutsumi, Nobuhiro; Isobe, Keisuke; Itoh, Kazuyoshi

2007-03-01

Spatial and temporal information about intracellular objects and their dynamics within a living cell are essential for dynamic analysis of such objects in cell biology. A specific intracellular object can be discriminated by photoactivatable fluorescent proteins that exhibit pronounced light-induced spectral changes. Here, we report on selective labeling and tracking of a single organelle by using two-photon conversion of a photoconvertible fluorescent protein with near-infrared femtosecond laser pulses. We performed selective labeling of a single mitochondrion in a living tobacco BY-2 cell using two-photon photoconversion of Kaede. Using this technique, we demonstrated that, in plants, the directed movement of individual mitochondria along the cytoskeletons was mediated by actin filaments, whereas microtubules were not required for the movement of mitochondria. This single-organelle labeling technique enabled us to track the dynamics of a single organelle, revealing the mechanisms involved in organelle dynamics. The technique has potential application in direct tracking of selective cellular and intracellular structures.

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

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.

Millisecond single-molecule localization microscopy combined with convolution analysis and automated image segmentation to determine protein concentrations in complexly structured, functional cells, one cell at a time.

Science.gov (United States)

Wollman, Adam J M; Leake, Mark C

2015-01-01

We present a single-molecule tool called the CoPro (concentration of proteins) method that uses millisecond imaging with convolution analysis, automated image segmentation and super-resolution localization microscopy to generate robust estimates for protein concentration in different compartments of single living cells, validated using realistic simulations of complex multiple compartment cell types. We demonstrate its utility experimentally on model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast cells, and use it to address the biological question of how signals are transduced in cells. Cells in all domains of life dynamically sense their environment through signal transduction mechanisms, many involving gene regulation. The glucose sensing mechanism of S. cerevisiae is a model system for studying gene regulatory signal transduction. It uses the multi-copy expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in the presence of elevated extracellular glucose concentrations. We fluorescently labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal integration at physiological expression levels in living S. cerevisiae cells, in addition to the RNA polymerase protein Nrd1 with the fluorescent protein reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1 protein concentrations in the cytoplasm and nucleus compartments on a cell-by-cell basis under physiological conditions. These estimates indicate a ∼4-fold shift towards higher values in the concentration of diffusive Mig1 in the nucleus if the external glucose concentration is raised, whereas equivalent levels in the cytoplasm shift to smaller values with a relative change an order of magnitude smaller. This compares with Nrd1 which is not involved directly in glucose sensing, and which is almost exclusively localized in the nucleus under high and low external glucose levels. CoPro facilitates time-resolved quantification of

Model-based design of RNA hybridization networks implemented in living cells.

Science.gov (United States)

Rodrigo, Guillermo; Prakash, Satya; Shen, Shensi; Majer, Eszter; Daròs, José-Antonio; Jaramillo, Alfonso

2017-09-19

Synthetic gene circuits allow the behavior of living cells to be reprogrammed, and non-coding small RNAs (sRNAs) are increasingly being used as programmable regulators of gene expression. However, sRNAs (natural or synthetic) are generally used to regulate single target genes, while complex dynamic behaviors would require networks of sRNAs regulating each other. Here, we report a strategy for implementing such networks that exploits hybridization reactions carried out exclusively by multifaceted sRNAs that are both targets of and triggers for other sRNAs. These networks are ultimately coupled to the control of gene expression. We relied on a thermodynamic model of the different stable conformational states underlying this system at the nucleotide level. To test our model, we designed five different RNA hybridization networks with a linear architecture, and we implemented them in Escherichia coli. We validated the network architecture at the molecular level by native polyacrylamide gel electrophoresis, as well as the network function at the bacterial population and single-cell levels with a fluorescent reporter. Our results suggest that it is possible to engineer complex cellular programs based on RNA from first principles. Because these networks are mainly based on physical interactions, our designs could be expanded to other organisms as portable regulatory resources or to implement biological computations. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

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)

Single cell time-lapse analysis reveals that podoplanin enhances cell survival and colony formation capacity of squamous cell carcinoma cells.

Science.gov (United States)

Miyashita, Tomoyuki; Higuchi, Youichi; Kojima, Motohiro; Ochiai, Atsushi; Ishii, Genichiro

2017-01-06

Tumor initiating cells (TICs) are characterized by high clonal expansion capacity. We previously reported that podoplanin is a TIC-specific marker for the human squamous cell carcinoma cell line A431. The aim of this study is to explore the molecular mechanism underlying the high clonal expansion potential of podoplanin-positive A431cells using Fucci imaging. Single podoplanin-positive cells created large colonies at a significantly higher frequency than single podoplanin-negative cells, whereas no difference was observed between the two types of cells with respect to cell cycle status. Conversely, the cell death ratio of progenies derived from podoplanin-positive single cell was significantly lower than that of cells derived from podoplanin-negative cells. Single A431 cells, whose podoplanin expression was suppressed by RNA interference, exhibited increased cell death ratios and decreased frequency of large colony forming. Moreover, the frequency of large colony forming decreased significantly when podoplanin-positive single cells was treated with a ROCK (Rho-associated coiled-coil kinase) inhibitor, whereas no difference was observed in single podoplanin-negative cells. Our current study cleared that high clonal expansion capacity of podoplanin-positive TICs populations was the result of reduced cell death by podoplanin-mediated signaling. Therefore, podoplanin activity may be a therapeutic target in the treatment of squamous cell carcinomas.

Quantifying Force and Viscoelasticity Inside Living Cells Using an Active–Passive Calibrated Optical Trap

DEFF Research Database (Denmark)

Ritter, Christine M.; Maes, Josep; Oddershede, Lene

2017-01-01

As described in the previous chapters, optical tweezers have become a tool of precision for in vitro single-molecule investigations, where the single molecule of interest most often is studied in purified form in an experimental assay with a well-controlled fluidic environment. A well-controlled ...... is that the size and refractive properties of the trapped object and the viscoelastic properties of its environment need not be known. We explain the protocol and demonstrate its use with experiments of trapped granules inside live S.pombe cells.......As described in the previous chapters, optical tweezers have become a tool of precision for in vitro single-molecule investigations, where the single molecule of interest most often is studied in purified form in an experimental assay with a well-controlled fluidic environment. A well......-controlled fluidic environment implies that the physical properties of the liquid, most notably the viscosity, are known and the fluidic environment can, for calibrational purposes, be treated as a simple liquid. In vivo, however, optical tweezers have primarily been used as a tool of manipulation and not so often...

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.

Quantum measurement and orientation tracking of fluorescent nanodiamonds inside living cells

Science.gov (United States)

McGuinness, L. P.; Yan, Y.; Stacey, A.; Simpson, D. A.; Hall, L. T.; MacLaurin, D.; Prawer, S.; Mulvaney, P.; Wrachtrup, J.; Caruso, F.; Scholten, R. E.; Hollenberg, L. C. L.

2011-06-01

Fluorescent particles are routinely used to probe biological processes. The quantum properties of single spins within fluorescent particles have been explored in the field of nanoscale magnetometry, but not yet in biological environments. Here, we demonstrate optically detected magnetic resonance of individual fluorescent nanodiamond nitrogen-vacancy centres inside living human HeLa cells, and measure their location, orientation, spin levels and spin coherence times with nanoscale precision. Quantum coherence was measured through Rabi and spin-echo sequences over long (>10 h) periods, and orientation was tracked with effective 1° angular precision over acquisition times of 89 ms. The quantum spin levels served as fingerprints, allowing individual centres with identical fluorescence to be identified and tracked simultaneously. Furthermore, monitoring decoherence rates in response to changes in the local environment may provide new information about intracellular processes. The experiments reported here demonstrate the viability of controlled single spin probes for nanomagnetometry in biological systems, opening up a host of new possibilities for quantum-based imaging in the life sciences.

Biological aspects of the development and self-concept in adolescents living in single-parent families.

Science.gov (United States)

Vecek, Andrea; Vidović, Vesna; Milicić, Jasna; Spoljar-Vrzina, Sanja; Vecek, Nenad; Arch-Vecek, Branka

2009-09-01

In this study we investigate whether there are differences between adolescents who grow up in single-parent families and those who grow up in nucleus families. We have decided that there are no differences in the physical development between the adolescents who are growing up in single parent families and those growing up in nucleus families. There is no difference in the self-concept between these two groups, except in the ethical and moral self-image of adolescents living with one parent. Adolescents living in single-parent families have a weaker moral self-image. It can thus be concluded that physical development and positive self-concept (a favorable image of oneself) in adolescents do not depend on whether an adolescent is growing up in a single-parent or a nucleus family, but on the different characteristics of parents and their relationship with children, whether they are married or not. For the children development the best is healthy marriage of their parents.

MICROORGANISMS: A MARVELOUS SOURCE OF SINGLE CELL PROTEINS

Directory of Open Access Journals (Sweden)

Agam Nangul

2013-08-01

Full Text Available The increasing global population living below the poverty line is driving the scientific community to search for non-conventional protein sources that can replace conventional expensive ones. Microbial proteins, or single-cell protein (SCP, represent a potential future nutrient source for human food and animal feed. These microbial proteins can be grown rapidly on substrates with minimum dependence on soil, water and climate conditions. They can be produced from algae, fungi and bacteria the chief sources of SCP. It is convenient to use microorganisms for production of SCP as they grow rapidly and have high protein content. Industrially, they can be produced from algal biomass, yeast, fungi. There are several other ways of getting SCP as well. Despite numerous advantages of SCP, they have disadvantages and toxic effects too, especially related to mycotoxins and bacterial toxins.

Single cell Hi-C reveals cell-to-cell variability in chromosome structure

Science.gov (United States)

Schoenfelder, Stefan; Yaffe, Eitan; Dean, Wendy; Laue, Ernest D.; Tanay, Amos; Fraser, Peter

2013-01-01

Large-scale chromosome structure and spatial nuclear arrangement have been linked to control of gene expression and DNA replication and repair. Genomic techniques based on chromosome conformation capture assess contacts for millions of loci simultaneously, but do so by averaging chromosome conformations from millions of nuclei. Here we introduce single cell Hi-C, combined with genome-wide statistical analysis and structural modeling of single copy X chromosomes, to show that individual chromosomes maintain domain organisation at the megabase scale, but show variable cell-to-cell chromosome territory structures at larger scales. Despite this structural stochasticity, localisation of active gene domains to boundaries of territories is a hallmark of chromosomal conformation. Single cell Hi-C data bridge current gaps between genomics and microscopy studies of chromosomes, demonstrating how modular organisation underlies dynamic chromosome structure, and how this structure is probabilistically linked with genome activity patterns. PMID:24067610

Capturing Three-Dimensional Genome Organization in Individual Cells by Single-Cell Hi-C.

Science.gov (United States)

Nagano, Takashi; Wingett, Steven W; Fraser, Peter

2017-01-01

Hi-C is a powerful method to investigate genome-wide, higher-order chromatin and chromosome conformations averaged from a population of cells. To expand the potential of Hi-C for single-cell analysis, we developed single-cell Hi-C. Similar to the existing "ensemble" Hi-C method, single-cell Hi-C detects proximity-dependent ligation events between cross-linked and restriction-digested chromatin fragments in cells. A major difference between the single-cell Hi-C and ensemble Hi-C protocol is that the proximity-dependent ligation is carried out in the nucleus. This allows the isolation of individual cells in which nearly the entire Hi-C procedure has been carried out, enabling the production of a Hi-C library and data from individual cells. With this new method, we studied genome conformations and found evidence for conserved topological domain organization from cell to cell, but highly variable interdomain contacts and chromosome folding genome wide. In addition, we found that the single-cell Hi-C protocol provided cleaner results with less technical noise suggesting it could be used to improve the ensemble Hi-C technique.

Avian metapneumovirus M2:2 protein inhibits replication in Vero cells: modification facilitates live vaccine development.

Science.gov (United States)

Clubbe, Jayne; Naylor, Clive J

2011-11-28

Throughout the world, avian metapneumovirus (AMPV) infection of subtype A is principally controlled by two live vaccines both derived from UK field strain #8544. Improvements of those vaccines by use of reverse genetics technology was found to be hampered by the inability of #8544 to replicate in the commonly exploited Vero cell based reverse genetics system. A systematic reverse genetics based genome modification of a DNA copy of #8544, employing sequence data from a Vero grown, #8544 derived, live vaccine; was used to determine mutations required to facilitate virus recovery and replication in Vero cells. This identified a single coding substitution in the M2:2 reading frame as responsible. Furthermore, ablation of M2:2 was found to elicit the same outcome. M2:2 sequence analysis of seven AMPVs found Vero cell adaption to be associated with non similar amino acid changes in M2:2. The study shows that M2:2 modification of field virus #8544 will enable research leading to improved vaccines. This may have more general application to other AMPV field strains. Copyright © 2011 Elsevier Ltd. All rights reserved.

mRNA-Seq of single prostate cancer circulating tumor cells reveals recapitulation of gene expression and pathways found in prostate cancer.

Directory of Open Access Journals (Sweden)

Gordon M Cann

Full Text Available Circulating tumor cells (CTC mediate metastatic spread of many solid tumors and enumeration of CTCs is currently used as a prognostic indicator of survival in metastatic prostate cancer patients. Some evidence suggests that it is possible to derive additional information about tumors from expression analysis of CTCs, but the technical difficulty of isolating and analyzing individual CTCs has limited progress in this area. To assess the ability of a new generation of MagSweeper to isolate intact CTCs for downstream analysis, we performed mRNA-Seq on single CTCs isolated from the blood of patients with metastatic prostate cancer and on single prostate cancer cell line LNCaP cells spiked into the blood of healthy donors. We found that the MagSweeper effectively isolated CTCs with a capture efficiency that matched the CellSearch platform. However, unlike CellSearch, the MagSweeper facilitates isolation of individual live CTCs without contaminating leukocytes. Importantly, mRNA-Seq analysis showed that the MagSweeper isolation process did not have a discernible impact on the transcriptional profile of single LNCaPs isolated from spiked human blood, suggesting that any perturbations caused by the MagSweeper process on the transcriptional signature of isolated cells are modest. Although the RNA from patient CTCs showed signs of significant degradation, consistent with reports of short half-lives and apoptosis amongst CTCs, transcriptional signatures of prostate tissue and of cancer were readily detectable with single CTC mRNA-Seq. These results demonstrate that the MagSweeper provides access to intact CTCs and that these CTCs can potentially supply clinically relevant information.

Single-cell photoacoustic thermometry

Science.gov (United States)

Gao, Liang; Wang, Lidai; Li, Chiye; Liu, Yan; Ke, Haixin; Zhang, Chi

2013-01-01

Abstract. A novel photoacoustic thermometric method is presented for simultaneously imaging cells and sensing their temperature. With three-seconds-per-frame imaging speed, a temperature resolution of 0.2°C was achieved in a photo-thermal cell heating experiment. Compared to other approaches, the photoacoustic thermometric method has the advantage of not requiring custom-developed temperature-sensitive biosensors. This feature should facilitate the conversion of single-cell thermometry into a routine lab tool and make it accessible to a much broader biological research community. PMID:23377004

Cell type discovery using single-cell transcriptomics: implications for ontological representation.

Science.gov (United States)

Aevermann, Brian D; Novotny, Mark; Bakken, Trygve; Miller, Jeremy A; Diehl, Alexander D; Osumi-Sutherland, David; Lasken, Roger S; Lein, Ed S; Scheuermann, Richard H

2018-05-01

Cells are fundamental function units of multicellular organisms, with different cell types playing distinct physiological roles in the body. The recent advent of single-cell transcriptional profiling using RNA sequencing is producing 'big data', enabling the identification of novel human cell types at an unprecedented rate. In this review, we summarize recent work characterizing cell types in the human central nervous and immune systems using single-cell and single-nuclei RNA sequencing, and discuss the implications that these discoveries are having on the representation of cell types in the reference Cell Ontology (CL). We propose a method, based on random forest machine learning, for identifying sets of necessary and sufficient marker genes, which can be used to assemble consistent and reproducible cell type definitions for incorporation into the CL. The representation of defined cell type classes and their relationships in the CL using this strategy will make the cell type classes being identified by high-throughput/high-content technologies findable, accessible, interoperable and reusable (FAIR), allowing the CL to serve as a reference knowledgebase of information about the role that distinct cellular phenotypes play in human health and disease.

A single-cell resolution map of mouse hematopoietic stem and progenitor cell differentiation.

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Nestorowa, Sonia; Hamey, Fiona K; Pijuan Sala, Blanca; Diamanti, Evangelia; Shepherd, Mairi; Laurenti, Elisa; Wilson, Nicola K; Kent, David G; Göttgens, Berthold

2016-08-25

Maintenance of the blood system requires balanced cell fate decisions by hematopoietic stem and progenitor cells (HSPCs). Because cell fate choices are executed at the individual cell level, new single-cell profiling technologies offer exciting possibilities for mapping the dynamic molecular changes underlying HSPC differentiation. Here, we have used single-cell RNA sequencing to profile more than 1600 single HSPCs, and deep sequencing has enabled detection of an average of 6558 protein-coding genes per cell. Index sorting, in combination with broad sorting gates, allowed us to retrospectively assign cells to 12 commonly sorted HSPC phenotypes while also capturing intermediate cells typically excluded by conventional gating. We further show that independently generated single-cell data sets can be projected onto the single-cell resolution expression map to directly compare data from multiple groups and to build and refine new hypotheses. Reconstruction of differentiation trajectories reveals dynamic expression changes associated with early lymphoid, erythroid, and granulocyte-macrophage differentiation. The latter two trajectories were characterized by common upregulation of cell cycle and oxidative phosphorylation transcriptional programs. By using external spike-in controls, we estimate absolute messenger RNA (mRNA) levels per cell, showing for the first time that despite a general reduction in total mRNA, a subset of genes shows higher expression levels in immature stem cells consistent with active maintenance of the stem-cell state. Finally, we report the development of an intuitive Web interface as a new community resource to permit visualization of gene expression in HSPCs at single-cell resolution for any gene of choice. © 2016 by The American Society of Hematology.

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

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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.

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.

Single-cell and population NF-κB dynamic responses depend on lipopolysaccharide preparation.

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Miriam V Gutschow

Full Text Available Lipopolysaccharide (LPS, found in the outer membrane of gram-negative bacteria, elicits a strong response from the transcription factor family Nuclear factor (NF-κB via Toll-like receptor (TLR 4. The cellular response to lipopolysaccharide varies depending on the source and preparation of the ligand, however. Our goal was to compare single-cell NF-κB dynamics across multiple sources and concentrations of LPS.Using live-cell fluorescence microscopy, we determined the NF-κB activation dynamics of hundreds of single cells expressing a p65-dsRed fusion protein. We used computational image analysis to measure the nuclear localization of the fusion protein in the cells over time. The concentration range spanned up to nine orders of magnitude for three E. coli LPS preparations. We find that the LPS preparations induce markedly different responses, even accounting for potency differences. We also find that the ability of soluble TNF receptor to affect NF-κB dynamics varies strikingly across the three preparations.Our work strongly suggests that the cellular response to LPS is highly sensitive to the source and preparation of the ligand. We therefore caution that conclusions drawn from experiments using one preparation may not be applicable to LPS in general.

Fibered confocal fluorescence microscopy for imaging apoptotic DNA fragmentation at the single-cell level in vivo

International Nuclear Information System (INIS)

Al-Gubory, Kais H.

2005-01-01

The major characteristic of cell death by apoptosis is the loss of nuclear DNA integrity by endonucleases, resulting in the formation of small DNA fragments. The application of confocal imaging to in vivo monitoring of dynamic cellular events, like apoptosis, within internal organs and tissues has been limited by the accessibility to these sites. Therefore, the aim of the present study was to test the feasibility of fibered confocal fluorescence microscopy (FCFM) to image in situ apoptotic DNA fragmentation in surgically exteriorized sheep corpus luteum in the living animal. Following intra-luteal administration of a fluorescent DNA-staining dye, YO-PRO-1, DNA cleavage within nuclei of apoptotic cells was serially imaged at the single-cell level by FCFM. This imaging technology is sufficiently simple and rapid to allow time series in situ detection and visualization of cells undergoing apoptosis in the intact animal. Combined with endoscope, this approach can be used for minimally invasive detection of fluorescent signals and visualization of cellular events within internal organs and tissues and thereby provides the opportunity to study biological processes in the natural physiological environment of the cell in living animals

Cloning of Plasmodium falciparum by single-cell sorting.

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Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-10-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two-dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. Copyright 2010 Elsevier Inc. All rights reserved.

Cloning of Plasmodium falciparum by single-cell sorting

Science.gov (United States)

Miao, Jun; Li, Xiaolian; Cui, Liwang

2010-01-01

Malaria parasite cloning is traditionally carried out mainly by using the limiting dilution method, which is laborious, imprecise, and unable to distinguish multiply-infected RBCs. In this study, we used a parasite engineered to express green fluorescent protein (GFP) to evaluate a single-cell sorting method for rapidly cloning Plasmodium falciparum. By dividing a two dimensional scattergram from a cell sorter into 17 gates, we determined the parameters for isolating singly-infected erythrocytes and sorted them into individual cultures. Pre-gating of the engineered parasites for GFP allowed the isolation of almost 100% GFP-positive clones. Compared with the limiting dilution method, the number of parasite clones obtained by single-cell sorting was much higher. Molecular analyses showed that parasite isolates obtained by single-cell sorting were highly homogenous. This highly efficient single-cell sorting method should prove very useful for cloning both P. falciparum laboratory populations from genetic manipulation experiments and clinical samples. PMID:20435038

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.

Single-Cell Quantitative PCR: Advances and Potential in Cancer Diagnostics.

Science.gov (United States)

Ok, Chi Young; Singh, Rajesh R; Salim, Alaa A

2016-01-01

Tissues are heterogeneous in their components. If cells of interest are a minor population of collected tissue, it would be difficult to obtain genetic or genomic information of the interested cell population with conventional genomic DNA extraction from the collected tissue. Single-cell DNA analysis is important in the analysis of genetics of cell clonality, genetic anticipation, and single-cell DNA polymorphisms. Single-cell PCR using Single Cell Ampligrid/GeXP platform is described in this chapter.

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

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.

A quantitative method to track protein translocation between intracellular compartments in real-time in live cells using weighted local variance image analysis.

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Guillaume Calmettes

Full Text Available The genetic expression of cloned fluorescent proteins coupled to time-lapse fluorescence microscopy has opened the door to the direct visualization of a wide range of molecular interactions in living cells. In particular, the dynamic translocation of proteins can now be explored in real time at the single-cell level. Here we propose a reliable, easy-to-implement, quantitative image processing method to assess protein translocation in living cells based on the computation of spatial variance maps of time-lapse images. The method is first illustrated and validated on simulated images of a fluorescently-labeled protein translocating from mitochondria to cytoplasm, and then applied to experimental data obtained with fluorescently-labeled hexokinase 2 in different cell types imaged by regular or confocal microscopy. The method was found to be robust with respect to cell morphology changes and mitochondrial dynamics (fusion, fission, movement during the time-lapse imaging. Its ease of implementation should facilitate its application to a broad spectrum of time-lapse imaging studies.

Droplet Microfluidics for Compartmentalized Cell Lysis and Extension of DNA from Single-Cells

Science.gov (United States)

Zimny, Philip; Juncker, David; Reisner, Walter

Current single cell DNA analysis methods suffer from (i) bias introduced by the need for molecular amplification and (ii) limited ability to sequence repetitive elements, resulting in (iii) an inability to obtain information regarding long range genomic features. Recent efforts to circumvent these limitations rely on techniques for sensing single molecules of DNA extracted from single-cells. Here we demonstrate a droplet microfluidic approach for encapsulation and biochemical processing of single-cells inside alginate microparticles. In our approach, single-cells are first packaged inside the alginate microparticles followed by cell lysis, DNA purification, and labeling steps performed off-chip inside this microparticle system. The alginate microparticles are then introduced inside a micro/nanofluidic system where the alginate is broken down via a chelating buffer, releasing long DNA molecules which are then extended inside nanofluidic channels for analysis via standard mapping protocols.

Simultaneous Measurement of Multiple Mechanical Properties of Single Cells Using AFM by Indentation and Vibration.

Science.gov (United States)

Zhang, Chuang; Shi, Jialin; Wang, Wenxue; Xi, Ning; Wang, Yuechao; Liu, Lianqing

2017-12-01

The mechanical properties of cells, which are the main characteristics determining their physical performance and physiological functions, have been actively studied in the fields of cytobiology and biomedical engineering and for the development of medicines. In this study, an indentation-vibration-based method is proposed to simultaneously measure the mechanical properties of cells in situ, including cellular mass (m), elasticity (k), and viscosity (c). The proposed measurement method is implemented based on the principle of forced vibration stimulated by simple harmonic force using an atomic force microscope (AFM) system integrated with a piezoelectric transducer as the substrate vibrator. The corresponding theoretical model containing the three mechanical properties is derived and used to perform simulations and calculations. Living and fixed human embryonic kidney 293 (HEK 293) cells were subjected to indentation and vibration to measure and compare their mechanical parameters and verify the proposed approach. The results that the fixed sample cells are more viscous and elastic than the living sample cells and the measured mechanical properties of cell are consistent within, but not outside of the central region of the cell, are in accordance with the previous studies. This work provides an approach to simultaneous measurement of the multiple mechanical properties of single cells using an integrated AFM system based on the principle force vibration and thickness-corrected Hertz model. This study should contribute to progress in biomedical engineering, cytobiology, medicine, early diagnosis, specific therapy and cell-powered robots.

Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells

International Nuclear Information System (INIS)

Gedik, C.M.; Collins, A.R.; Ewen, S.W.B.

1992-01-01

The authors have adapted procedure of single cell gel electrophoresis (SCGE) for studying DNA damage and repair induced by UV-C-radiation, using HeLa cells. UV-C itself does not induce DNA breakage, and though cellular repair of UV-C damage produces DNA breaks as intermediates, these are too short-lived to be detected by SCGE. Incubation of UV-C-irradiated cells with the DNA synthesis inhibitor aphidicolin causes accumulation of incomplete repair sites to a level readily detected by SCGE even after doses as low as 0.5 J m -2 and incubation for as little as 5 min. The authors also studied UV-C-dependent incision, repair synthesis and ligation in permeable cells. Finally, key incubated permeable cells, after UV-C-irradiation, with exogenous UV endonuclease, examined consequent breaks both by SCGE and by alkaline unwinding to express results of the electrophoretic method in terms of DNA break frequencies. The sensitivity of the SCGE technique can thus be estimated; as few as 0.1 DNA breaks per 10 9 daltons are detected. (Author)

Mapping Cellular Hierarchy by Single-Cell Analysis of the Cell Surface Repertoire

OpenAIRE

Guo, Guoji; Luc, Sidinh; Marco, Eugenio; Lin, Ta-Wei; Peng, Cong; Kerenyi, Marc A.; Beyaz, Semir; Kim, Woojin; Xu, Jian; Das, Partha Pratim; Neff, Tobias; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H.

2013-01-01

Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method we analyzed over 1500 single cells throughout the mouse hematopoietic system, and illustrate its utility for revealing important biological insi...

Live Cells Decreased Methane Production in Intestinal Content of Pigs

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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

Live-cell imaging: new avenues to investigate retinal regeneration

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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.

Genomic Sequencing of Single Microbial Cells from Environmental Samples

Energy Technology Data Exchange (ETDEWEB)

Ishoey, Thomas; Woyke, Tanja; Stepanauskas, Ramunas; Novotny, Mark; Lasken, Roger S.

2008-02-01

Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification, Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.

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.