Manipulating motions of targeted single cells in solution by an integrated double-ring magnetic tweezers imaging microscope

NASA Astrophysics Data System (ADS)

Wu, Meiling; Yadav, Rajeev; Pal, Nibedita; Lu, H. Peter

2017-07-01

Controlling and manipulating living cell motions in solution hold a high promise in developing new biotechnology and biological science. Here, we developed a magnetic tweezers device that employs a combination of two permanent magnets in up-down double-ring configuration axially fitting with a microscopic objective, allowing a picoNewton (pN) bidirectional force and motion control on the sample beyond a single upward pulling direction. The experimental force calibration and magnetic field simulation using finite element method magnetics demonstrate that the designed magnetic tweezers covers a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve motion manipulation by shifting the force balance. We demonstrate an application of the up-down double-ring magnetic tweezers for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion.

Optoelectronic tweezers integrated with lensfree holographic microscopy for wide-field interactive cell and particle manipulation on a chip.

PubMed

Huang, Kuo-Wei; Su, Ting-Wei; Ozcan, Aydogan; Chiou, Pei-Yu

2013-06-21

We demonstrate an optoelectronic tweezer (OET) coupled to a lensfree holographic microscope for real-time interactive manipulation of cells and micro-particles over a large field-of-view (FOV). This integrated platform can record the holographic images of cells and particles over the entire active area of a CCD sensor array, perform digital image reconstruction to identify target cells, dynamically track the positions of cells and particles, and project light beams to trigger light-induced dielectrophoretic forces to pattern and sort cells on a chip. OET technology has been previously shown to be capable of performing parallel single cell manipulation over a large area. However, its throughput has been bottlenecked by the number of cells that can be imaged within the limited FOV of a conventional microscope objective lens. Integrating lensfree holographic imaging with OET solves this fundamental FOV barrier, while also creating a compact on-chip cell/particle manipulation platform. Using this unique platform, we have successfully demonstrated real-time interactive manipulation of thousands of single cells and micro-particles over an ultra-large area of e.g., 240 mm(2) (i.e. 17.96 mm × 13.52 mm).

Laser Nano-Neurosurgery from Gentle Manipulation to Nano-Incision of Neuronal Cells and Scaffolds: An Advanced Neurotechnology Tool.

PubMed

Soloperto, Alessandro; Palazzolo, Gemma; Tsushima, Hanako; Chieregatti, Evelina; Vassalli, Massimo; Difato, Francesco

2016-01-01

Current optical approaches are progressing far beyond the scope of monitoring the structure and function of living matter, and they are becoming widely recognized as extremely precise, minimally-invasive, contact-free handling tools. Laser manipulation of living tissues, single cells, or even single-molecules is becoming a well-established methodology, thus founding the onset of new experimental paradigms and research fields. Indeed, a tightly focused pulsed laser source permits complex tasks such as developing engineered bioscaffolds, applying calibrated forces, transfecting, stimulating, or even ablating single cells with subcellular precision, and operating intracellular surgical protocols at the level of single organelles. In the present review, we report the state of the art of laser manipulation in neuroscience, to inspire future applications of light-assisted tools in nano-neurosurgery.

Laser Nano-Neurosurgery from Gentle Manipulation to Nano-Incision of Neuronal Cells and Scaffolds: An Advanced Neurotechnology Tool

PubMed Central

Soloperto, Alessandro; Palazzolo, Gemma; Tsushima, Hanako; Chieregatti, Evelina; Vassalli, Massimo; Difato, Francesco

2016-01-01

Current optical approaches are progressing far beyond the scope of monitoring the structure and function of living matter, and they are becoming widely recognized as extremely precise, minimally-invasive, contact-free handling tools. Laser manipulation of living tissues, single cells, or even single-molecules is becoming a well-established methodology, thus founding the onset of new experimental paradigms and research fields. Indeed, a tightly focused pulsed laser source permits complex tasks such as developing engineered bioscaffolds, applying calibrated forces, transfecting, stimulating, or even ablating single cells with subcellular precision, and operating intracellular surgical protocols at the level of single organelles. In the present review, we report the state of the art of laser manipulation in neuroscience, to inspire future applications of light-assisted tools in nano-neurosurgery. PMID:27013962

Single-cell-based breeding: Rational strategy for the establishment of cell lines from a single cell with the most favorable properties.

PubMed

Yoshimoto, Nobuo; Kuroda, Shun'ichi

2014-04-01

For efficient biomolecule production (e.g., antibodies, recombinant proteins), mammalian cells with high expression rates should be selected from cell libraries, propagated while maintaining a homogenous expression rate, and subsequently stabilized at their high expression rate. Clusters of isogenic cells (i.e., colonies) have been used for these processes. However, cellular heterogeneity makes it difficult to obtain cell lines with the highest expression rates by using single-colony-based breeding. Furthermore, even among the single cells in an isogenic cell population, the desired cell properties fluctuate stochastically during long-term culture. Therefore, although the molecular mechanisms underlying stochastic fluctuation are poorly understood, it is necessary to establish excellent cell lines in order to breed single cells to have higher expression, higher stability, and higher homogeneity while suppressing stochastic fluctuation (i.e., single-cell-based breeding). In this review, we describe various methods for manipulating single cells and facilitating single-cell analysis in order to better understand stochastic fluctuation. We demonstrated that single-cell-based breeding is practical and promising by using a high-throughput automated system to analyze and manipulate single cells. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

MEMS-based platforms for mechanical manipulation and characterization of cells

NASA Astrophysics Data System (ADS)

Pan, Peng; Wang, Wenhui; Ru, Changhai; Sun, Yu; Liu, Xinyu

2017-12-01

Mechanical manipulation and characterization of single cells are important experimental techniques in biological and medical research. Because of the microscale sizes and highly fragile structures of cells, conventional cell manipulation and characterization techniques are not accurate and/or efficient enough or even cannot meet the more and more demanding needs in different types of cell-based studies. To this end, novel microelectromechanical systems (MEMS)-based technologies have been developed to improve the accuracy, efficiency, and consistency of various cell manipulation and characterization tasks, and enable new types of cell research. This article summarizes existing MEMS-based platforms developed for cell mechanical manipulation and characterization, highlights their specific design considerations making them suitable for their designated tasks, and discuss their advantages and limitations. In closing, an outlook into future trends is also provided.

Manipulation of Suspended Single Cells by Microfluidics and Optical Tweezers

PubMed Central

Nève, Nathalie; Kohles, Sean S.; Winn, Shelley R.; Tretheway, Derek C.

2010-01-01

Chondrocytes and osteoblasts experience multiple stresses in vivo. The optimum mechanical conditions for cell health are not fully understood. This paper describes the optical and microfluidic mechanical manipulation of single suspended cells enabled by the μPIVOT, an integrated micron resolution particle image velocimeter (μPIV) and dual optical tweezers instrument (OT). In this study, we examine the viability and trap stiffness of cartilage cells, identify the maximum fluid-induced stresses possible in uniform and extensional flows, and compare the deformation characteristics of bone and muscle cells. These results indicate cell photodamage of chondrocytes is negligible for at least 20 min for laser powers below 30 mW, a dead cell presents less resistance to internal organelle rearrangement and deforms globally more than a viable cell, the maximum fluid-induced shear stresses are limited to ~15 mPa for uniform flows but may exceed 1 Pa for extensional flows, and osteoblasts show no deformation for shear stresses up to 250 mPa while myoblasts are more easily deformed and exhibit a modulated response to increasing stress. This suggests that global and/or local stresses can be applied to single cells without physical contact. Coupled with microfluidic sensors, these manipulations may provide unique methods to explore single cell biomechanics. PMID:20824110

Selective microrobot control using a thermally responsive microclamper for microparticle manipulation

NASA Astrophysics Data System (ADS)

Go, Gwangjun; Choi, Hyunchul; Jeong, Semi; Ko, Seong Young; Park, Jong-Oh; Park, Sukho

2016-03-01

Microparticle manipulation using a microrobot in an enclosed environment, such as a lab-on-a-chip, has been actively studied because an electromagnetic actuated microrobot can have accurate motility and wireless controllability. In most studies on electromagnetic actuated microrobots, only a single microrobot has been used to manipulate cells or microparticles. However, the use of a single microrobot can pose several limitations when performing multiple roles in microparticle manipulation. To overcome the limitations associated with using a single microrobot, we propose a new method for the control of multiple microrobots. Multiple microrobots can be controlled independently by an electromagnetic actuation system and multiple microclampers combined with microheaters. To select a specific microrobot among multiple microrobots, we propose a microclamper composed of a clamper structure using thermally responsive hydrogel and a microheater for controlling the microclamper. A fundamental test of the proposed microparticle manipulation system is performed by selecting a specific microrobot among multiple microrobots. Through the independent locomotion of multiple microrobots with U- and V-shaped tips, heterogeneous microparticle manipulation is demonstrated in the creation of a two-dimensional structure. In the future, our proposed multiple-microrobot system can be applied to tasks that are difficult to perform using a single microrobot, such as cell manipulation, cargo delivery, tissue assembly, and cloning.

Electric and Magnetic Manipulation of Biological Systems

NASA Astrophysics Data System (ADS)

Lee, H.; Hunt, T. P.; Liu, Y.; Ham, D.; Westervelt, R. M.

2005-06-01

New types of biological cell manipulation systems, a micropost matrix, a microelectromagnet matrix, and a microcoil array, were developed. The micropost matrix consists of post-shaped electrodes embedded in an insulating layer. With a separate ac voltage applied to each electrode, the micropost matrix generates dielectrophoretic force to trap and move individual biological cells. The microelectromagnet matrix consists of two arrays of straight wires aligned perpendicular to each other, that are covered with insulating layers. By independently controlling the current in each wire, the microelectromagnet matrix creates versatile magnetic fields to manipulate individual biological cells attached to magnetic beads. The microcoil array is a set of coils implemented in a foundry using a standard silicon fabrication technology. Current sources to the coils, and control circuits are integrated on a single chip, making the device self-contained. Versatile manipulation of biological cells was demonstrated using these devices by generating optimized electric or magnetic field patterns. A single yeast cell was trapped and positioned with microscopic resolution, and multiple yeast cells were trapped and independently moved along the separate paths for cell-sorting.

Cell manipulation in microfluidics.

PubMed

Yun, Hoyoung; Kim, Kisoo; Lee, Won Gu

2013-06-01

Recent advances in the lab-on-a-chip field in association with nano/microfluidics have been made for new applications and functionalities to the fields of molecular biology, genetic analysis and proteomics, enabling the expansion of the cell biology field. Specifically, microfluidics has provided promising tools for enhancing cell biological research, since it has the ability to precisely control the cellular environment, to easily mimic heterogeneous cellular environment by multiplexing, and to analyze sub-cellular information by high-contents screening assays at the single-cell level. Various cell manipulation techniques in microfluidics have been developed in accordance with specific objectives and applications. In this review, we examine the latest achievements of cell manipulation techniques in microfluidics by categorizing externally applied forces for manipulation: (i) optical, (ii) magnetic, (iii) electrical, (iv) mechanical and (v) other manipulations. We furthermore focus on history where the manipulation techniques originate and also discuss future perspectives with key examples where available.

Cell Signaling Experiments Driven by Optical Manipulation

PubMed Central

Difato, Francesco; Pinato, Giulietta; Cojoc, Dan

2013-01-01

Cell signaling involves complex transduction mechanisms in which information released by nearby cells or extracellular cues are transmitted to the cell, regulating fundamental cellular activities. Understanding such mechanisms requires cell stimulation with precise control of low numbers of active molecules at high spatial and temporal resolution under physiological conditions. Optical manipulation techniques, such as optical tweezing, mechanical stress probing or nano-ablation, allow handling of probes and sub-cellular elements with nanometric and millisecond resolution. PicoNewton forces, such as those involved in cell motility or intracellular activity, can be measured with femtoNewton sensitivity while controlling the biochemical environment. Recent technical achievements in optical manipulation have new potentials, such as exploring the actions of individual molecules within living cells. Here, we review the progress in optical manipulation techniques for single-cell experiments, with a focus on force probing, cell mechanical stimulation and the local delivery of active molecules using optically manipulated micro-vectors and laser dissection. PMID:23698758

Development of an Integrated Chip for Automatic Tracking and Positioning Manipulation for Single Cell Lysis

PubMed Central

Young, Chao-Wang; Hsieh, Jia-Ling; Ay, Chyung

2012-01-01

This study adopted a microelectromechanical fabrication process to design a chip integrated with electroosmotic flow and dielectrophoresis force for single cell lysis. Human histiocytic lymphoma U937 cells were driven rapidly by electroosmotic flow and precisely moved to a specific area for cell lysis. By varying the frequency of AC power, 15 V AC at 1 MHz of frequency configuration achieved 100% cell lysing at the specific area. The integrated chip could successfully manipulate single cells to a specific position and lysis. The overall successful rate of cell tracking, positioning, and cell lysis is 80%. The average speed of cell driving was 17.74 μm/s. This technique will be developed for DNA extraction in biomolecular detection. It can simplify pre-treatment procedures for biotechnological analysis of samples. PMID:22736957

Development of an integrated chip for automatic tracking and positioning manipulation for single cell lysis.

PubMed

Young, Chao-Wang; Hsieh, Jia-Ling; Ay, Chyung

2012-01-01

This study adopted a microelectromechanical fabrication process to design a chip integrated with electroosmotic flow and dielectrophoresis force for single cell lysis. Human histiocytic lymphoma U937 cells were driven rapidly by electroosmotic flow and precisely moved to a specific area for cell lysis. By varying the frequency of AC power, 15 V AC at 1 MHz of frequency configuration achieved 100% cell lysing at the specific area. The integrated chip could successfully manipulate single cells to a specific position and lysis. The overall successful rate of cell tracking, positioning, and cell lysis is 80%. The average speed of cell driving was 17.74 μm/s. This technique will be developed for DNA extraction in biomolecular detection. It can simplify pre-treatment procedures for biotechnological analysis of samples.

Functionalized nanopipettes: toward label-free, single cell biosensors.

PubMed

Actis, Paolo; Mak, Andy C; Pourmand, Nader

2010-08-01

Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms.

Functionalized nanopipettes: toward label-free, single cell biosensors

PubMed Central

Actis, Paolo; Mak, Andy C.

2010-01-01

Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms. PMID:20730113

Voltage controlled nano-injection system for single-cell surgery

PubMed Central

Seger, R. Adam; Actis, Paolo; Penfold, Catherine; Maalouf, Michelle; Vilozny, Boaz; Pourmand, Nader

2015-01-01

Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes. PMID:22899383

Voltage controlled nano-injection system for single-cell surgery.

PubMed

Adam Seger, R; Actis, Paolo; Penfold, Catherine; Maalouf, Michelle; Vilozny, Boaz; Pourmand, Nader

2012-09-28

Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes.

Hydrogel Droplet Microfluidics for High-Throughput Single Molecule/Cell Analysis.

PubMed

Zhu, Zhi; Yang, Chaoyong James

2017-01-17

Heterogeneity among individual molecules and cells has posed significant challenges to traditional bulk assays, due to the assumption of average behavior, which would lose important biological information in heterogeneity and result in a misleading interpretation. Single molecule/cell analysis has become an important and emerging field in biological and biomedical research for insights into heterogeneity between large populations at high resolution. Compared with the ensemble bulk method, single molecule/cell analysis explores the information on time trajectories, conformational states, and interactions of individual molecules/cells, all key factors in the study of chemical and biological reaction pathways. Various powerful techniques have been developed for single molecule/cell analysis, including flow cytometry, atomic force microscopy, optical and magnetic tweezers, single-molecule fluorescence spectroscopy, and so forth. However, some of them have the low-throughput issue that has to analyze single molecules/cells one by one. Flow cytometry is a widely used high-throughput technique for single cell analysis but lacks the ability for intercellular interaction study and local environment control. Droplet microfluidics becomes attractive for single molecule/cell manipulation because single molecules/cells can be individually encased in monodisperse microdroplets, allowing high-throughput analysis and manipulation with precise control of the local environment. Moreover, hydrogels, cross-linked polymer networks that swell in the presence of water, have been introduced into droplet microfluidic systems as hydrogel droplet microfluidics. By replacing an aqueous phase with a monomer or polymer solution, hydrogel droplets can be generated on microfluidic chips for encapsulation of single molecules/cells according to the Poisson distribution. The sol-gel transition property endows the hydrogel droplets with new functionalities and diversified applications in single molecule/cell analysis. The hydrogel can act as a 3D cell culture matrix to mimic the extracellular environment for long-term single cell culture, which allows further heterogeneity study in proliferation, drug screening, and metastasis at the single-cell level. The sol-gel transition allows reactions in solution to be performed rapidly and efficiently with product storage in the gel for flexible downstream manipulation and analysis. More importantly, controllable sol-gel regulation provides a new way to maintain phenotype-genotype linkages in the hydrogel matrix for high throughput molecular evolution. In this Account, we will review the hydrogel droplet generation on microfluidics, single molecule/cell encapsulation in hydrogel droplets, as well as the progress made by our group and others in the application of hydrogel droplet microfluidics for single molecule/cell analysis, including single cell culture, single molecule/cell detection, single cell sequencing, and molecular evolution.

Force-controlled manipulation of single cells: from AFM to FluidFM.

PubMed

Guillaume-Gentil, Orane; Potthoff, Eva; Ossola, Dario; Franz, Clemens M; Zambelli, Tomaso; Vorholt, Julia A

2014-07-01

The ability to perturb individual cells and to obtain information at the single-cell level is of central importance for addressing numerous biological questions. Atomic force microscopy (AFM) offers great potential for this prospering field. Traditionally used as an imaging tool, more recent developments have extended the variety of cell-manipulation protocols. Fluidic force microscopy (FluidFM) combines AFM with microfluidics via microchanneled cantilevers with nano-sized apertures. The crucial element of the technology is the connection of the hollow cantilevers to a pressure controller, allowing their operation in liquid as force-controlled nanopipettes under optical control. Proof-of-concept studies demonstrated a broad spectrum of single-cell applications including isolation, deposition, adhesion and injection in a range of biological systems. Copyright © 2014 Elsevier Ltd. All rights reserved.

Three-dimensional manipulation of single cells using surface acoustic waves.

PubMed

Guo, Feng; Mao, Zhangming; Chen, Yuchao; Xie, Zhiwei; Lata, James P; Li, Peng; Ren, Liqiang; Liu, Jiayang; Yang, Jian; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2016-02-09

The ability of surface acoustic waves to trap and manipulate micrometer-scale particles and biological cells has led to many applications involving "acoustic tweezers" in biology, chemistry, engineering, and medicine. Here, we present 3D acoustic tweezers, which use surface acoustic waves to create 3D trapping nodes for the capture and manipulation of microparticles and cells along three mutually orthogonal axes. In this method, we use standing-wave phase shifts to move particles or cells in-plane, whereas the amplitude of acoustic vibrations is used to control particle motion along an orthogonal plane. We demonstrate, through controlled experiments guided by simulations, how acoustic vibrations result in micromanipulations in a microfluidic chamber by invoking physical principles that underlie the formation and regulation of complex, volumetric trapping nodes of particles and biological cells. We further show how 3D acoustic tweezers can be used to pick up, translate, and print single cells and cell assemblies to create 2D and 3D structures in a precise, noninvasive, label-free, and contact-free manner.

Using a nanopore for single molecule detection and single cell transfection.

PubMed

Nelson, Edward M; Kurz, Volker; Shim, Jiwook; Timp, Winston; Timp, Gregory

2012-07-07

We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.

Optoelectronic tweezers for microparticle and cell manipulation

NASA Technical Reports Server (NTRS)

Wu, Ming Chiang (Inventor); Chiou, Pei Yu (Inventor); Ohta, Aaron T. (Inventor)

2009-01-01

An optical image-driven light induced dielectrophoresis (DEP) apparatus and method are described which provide for the manipulation of particles or cells with a diameter on the order of 100 .mu.m or less. The apparatus is referred to as optoelectric tweezers (OET) and provides a number of advantages over conventional optical tweezers, in particular the ability to perform operations in parallel and over a large area without damage to living cells. The OET device generally comprises a planar liquid-filled structure having one or more portions which are photoconductive to convert incoming light to a change in the electric field pattern. The light patterns are dynamically generated to provide a number of manipulation structures that can manipulate single particles and cells or groups of particles/cells. The OET preferably includes a microscopic imaging means to provide feedback for the optical manipulation, such as detecting position and characteristics wherein the light patterns are modulated accordingly.

Optoelectronic Tweezers for Microparticle and Cell Manipulation

NASA Technical Reports Server (NTRS)

Wu, Ming Chiang (Inventor); Chiou, Pei-Yu (Inventor); Ohta, Aaron T. (Inventor)

2014-01-01

An optical image-driven light induced dielectrophoresis (DEP) apparatus and method are described which provide for the manipulation of particles or cells with a diameter on the order of 100 micromillimeters or less. The apparatus is referred to as optoelectric tweezers (OET) and provides a number of advantages over conventional optical tweezers, in particular the ability to perform operations in parallel and over a large area without damage to living cells. The OET device generally comprises a planar liquid-filled structure having one or more portions which are photoconductive to convert incoming light to a change in the electric field pattern. The light patterns are dynamically generated to provide a number of manipulation structures that can manipulate single particles and cells or group of particles/cells. The OET preferably includes a microscopic imaging means to provide feedback for the optical manipulation, such as detecting position and characteristics wherein the light patterns are modulated accordingly.

Magnetic domain wall conduits for single cell applications.

PubMed

Donolato, M; Torti, A; Kostesha, N; Deryabina, M; Sogne, E; Vavassori, P; Hansen, M F; Bertacco, R

2011-09-07

The ability to trap, manipulate and release single cells on a surface is important both for fundamental studies of cellular processes and for the development of novel lab-on-chip miniaturized tools for biological and medical applications. In this paper we demonstrate how magnetic domain walls generated in micro- and nano-structures fabricated on a chip surface can be used to handle single yeast cells labeled with magnetic beads. In detail, first we show that the proposed approach maintains the microorganism viable, as proven by monitoring the division of labeled yeast cells trapped by domain walls over 16 hours. Moreover, we demonstrate the controlled transport and release of individual yeast cells via displacement and annihilation of individual domain walls in micro- and nano-sized magnetic structures. These results pave the way to the implementation of magnetic devices based on domain walls technology in lab-on-chip systems devoted to accurate individual cell trapping and manipulation.

Determination of the forces imposed by micro and nanopipettes during DOPC: DOPS liposome manipulation.

PubMed

Allen, Kathleen B; Layton, Bradley E

2009-11-01

Using micropipette-based probing methods and an image processing algorithm for measuring deformation, the bending energies of aspirated DOPC:DOPS liposomes were estimated both before and during manipulation with an injection pipette. We found that unlike cells, which are penetrable with pipettes as large as 2mum in diameter and at speeds as slow as 4mum/s, liposomes, without a cytoskeleton to resist deformation, are impenetrable with pipettes as small as 25nm in diameter and at speeds as great as 4000mum/s. Using energy calculations and the previously published mechanical properties of DOPC:DOPS liposomes, the forces that injection pipettes of various sizes can exert onto liposomes during probing were estimated. Forces ranged from approximately 1pN to 6pN, and the forces exerted onto these liposomes increased as pipette size diminished. The quantification of the amount of force exerted on liposomes or cells during manipulation can assist in minimizing the damage during single-liposome, single-cell, or single-organelle injections and surgeries.

Review of methods to probe single cell metabolism and bioenergetics

DOE PAGES

Vasdekis, Andreas E.; Stephanopoulos, Gregory

2014-10-31

The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less

Endowing carbon nanotubes with superparamagnetic properties: applications for cell labeling, MRI cell tracking and magnetic manipulations.

PubMed

Lamanna, Giuseppe; Garofalo, Antonio; Popa, Gabriela; Wilhelm, Claire; Bégin-Colin, Sylvie; Felder-Flesch, Delphine; Bianco, Alberto; Gazeau, Florence; Ménard-Moyon, Cécilia

2013-05-21

Coating of carbon nanotubes (CNTs) with magnetic nanoparticles (NPs) imparts novel magnetic, optical, and thermal properties with potential applications in the biomedical domain. Multi-walled CNTs have been decorated with iron oxide superparamagnetic NPs. Two different approaches have been investigated based on ligand exchange or "click chemistry". The presence of the NPs on the nanotube surface allows conferring magnetic properties to CNTs. We have evaluated the potential of the NP/CNT hybrids as a contrast agent for magnetic resonance imaging (MRI) and their interactions with cells. The capacity of the hybrids to magnetically monitor and manipulate cells has also been investigated. The NP/CNTs can be manipulated by a remote magnetic field with enhanced contrast in MRI. They are internalized into tumor cells without showing cytotoxicity. The labeled cells can be magnetically manipulated as they display magnetic mobility and are detected at a single cell level through high resolution MRI.

Recent developments in microfluidics for cell studies.

PubMed

Xiong, Bin; Ren, Kangning; Shu, Yiwei; Chen, Yin; Shen, Bo; Wu, Hongkai

2014-08-20

As a technique for precisely manipulating fluid at the micrometer scale, the field of microfluidics has experienced an explosive growth over the past two decades, particularly owing to the advances in device design and fabrication. With the inherent advantages associated with its scale of operation, and its flexibility in being incorporated with other microscale techniques for manipulation and detection, microfluidics has become a major enabling technology, which has introduced new paradigms in various fields involving biological cells. A microfluidic device is able to realize functions that are not easily imaginable in conventional biological analysis, such as highly parallel, sophisticated high-throughput analysis, single-cell analysis in a well-defined manner, and tissue engineering with the capability of manipulation at the single-cell level. Major advancements in microfluidic device fabrication and the growing trend of implementing microfluidics in cell studies are presented, with a focus on biological research and clinical diagnostics. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis.

PubMed

Lo, Shih-Jie; Yao, Da-Jeng

2015-07-23

This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell.

Get to Understand More from Single-Cells: Current Studies of Microfluidic-Based Techniques for Single-Cell Analysis

PubMed Central

Lo, Shih-Jie; Yao, Da-Jeng

2015-01-01

This review describes the microfluidic techniques developed for the analysis of a single cell. The characteristics of microfluidic (e.g., little sample amount required, high-throughput performance) make this tool suitable to answer and to solve biological questions of interest about a single cell. This review aims to introduce microfluidic related techniques for the isolation, trapping and manipulation of a single cell. The major approaches for detection in single-cell analysis are introduced; the applications of single-cell analysis are then summarized. The review concludes with discussions of the future directions and opportunities of microfluidic systems applied in analysis of a single cell. PMID:26213918

Three-dimensional manipulation of single cells using surface acoustic waves

PubMed Central

Guo, Feng; Mao, Zhangming; Chen, Yuchao; Xie, Zhiwei; Lata, James P.; Li, Peng; Ren, Liqiang; Liu, Jiayang; Yang, Jian; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2016-01-01

The ability of surface acoustic waves to trap and manipulate micrometer-scale particles and biological cells has led to many applications involving “acoustic tweezers” in biology, chemistry, engineering, and medicine. Here, we present 3D acoustic tweezers, which use surface acoustic waves to create 3D trapping nodes for the capture and manipulation of microparticles and cells along three mutually orthogonal axes. In this method, we use standing-wave phase shifts to move particles or cells in-plane, whereas the amplitude of acoustic vibrations is used to control particle motion along an orthogonal plane. We demonstrate, through controlled experiments guided by simulations, how acoustic vibrations result in micromanipulations in a microfluidic chamber by invoking physical principles that underlie the formation and regulation of complex, volumetric trapping nodes of particles and biological cells. We further show how 3D acoustic tweezers can be used to pick up, translate, and print single cells and cell assemblies to create 2D and 3D structures in a precise, noninvasive, label-free, and contact-free manner. PMID:26811444

Three-dimensional holographic optical manipulation through a high-numerical-aperture soft-glass multimode fibre

NASA Astrophysics Data System (ADS)

Leite, Ivo T.; Turtaev, Sergey; Jiang, Xin; Šiler, Martin; Cuschieri, Alfred; Russell, Philip St. J.; Čižmár, Tomáš

2018-01-01

Holographic optical tweezers (HOT) hold great promise for many applications in biophotonics, allowing the creation and measurement of minuscule forces on biomolecules, molecular motors and cells. Geometries used in HOT currently rely on bulk optics, and their exploitation in vivo is compromised by the optically turbid nature of tissues. We present an alternative HOT approach in which multiple three-dimensional (3D) traps are introduced through a high-numerical-aperture multimode optical fibre, thus enabling an equally versatile means of manipulation through channels having cross-section comparable to the size of a single cell. Our work demonstrates real-time manipulation of 3D arrangements of micro-objects, as well as manipulation inside otherwise inaccessible cavities. We show that the traps can be formed over fibre lengths exceeding 100 mm and positioned with nanometric resolution. The results provide the basis for holographic manipulation and other high-numerical-aperture techniques, including advanced microscopy, through single-core-fibre endoscopes deep inside living tissues and other complex environments.

Single-cell trapping and selective treatment via co-flow within a microfluidic platform.

PubMed

Benavente-Babace, A; Gallego-Pérez, D; Hansford, D J; Arana, S; Pérez-Lorenzo, E; Mujika, M

2014-11-15

Lab on a chip (LOC) systems provide interesting and low-cost solutions for key studies and applications in the biomedical field. Along with microfluidics, these microdevices make single-cell manipulation possible with high spatial and temporal resolution. In this work we have designed, fabricated and characterized a versatile and inexpensive microfluidic platform for on-chip selective single-cell trapping and treatment using laminar co-flow. The combination of co-existing laminar flow manipulation and hydrodynamic single-cell trapping for selective treatment offers a cost-effective solution for studying the effect of novel drugs on single-cells. The operation of the whole system is experimentally simple, highly adaptable and requires no specific equipment. As a proof of concept, a cytotoxicity study of ethanol in isolated hepatocytes is presented. The developed microfluidic platform controlled by means of co-flow is an attractive and multipurpose solution for the study of new substances of high interest in cell biology research. In addition, this platform will pave the way for the study of cell behavior under dynamic and controllable fluidic conditions providing information at the individual cell level. Thus, this analysis device could also hold a great potential to easily use the trapped cells as sensing elements expanding its functionalities as a cell-based biosensor with single-cell resolution. Copyright © 2014 Elsevier B.V. All rights reserved.

Manipulation of biological cells using a microelectromagnet matrix

NASA Astrophysics Data System (ADS)

Lee, H.; Purdon, A. M.; Westervelt, R. M.

2004-08-01

Noninvasive manipulation of biological cells inside a microfluidic channel was demonstrated using a microelectromagnet matrix. The matrix consists of two layers of straight Au wires, aligned perpendicular to each other, that are covered by insulating layers. By adjusting the current in each independent wire, the microelectromagnet matrix can create versatile magnetic field patterns to control the motion of individual cells in fluid. Single or multiple yeast cells attached to magnetic beads were trapped, continuously moved and rotated, and a viable cell was separated from nonviable cells for cell sorting.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vasdekis, Andreas E.; Stephanopoulos, Gregory

The sampling and manipulation of cells down to the individual has been of substantial interest since the very beginning of Life Sciences. Herein, our objective is to highlight the most recent developments in single cell manipulation, as well as pioneering ones. First, flow-through methods will be discussed, namely methods in which the single cells flow continuously in an ordered manner during their analysis. This section will be followed by confinement techniques that enable cell isolation and confinement in one, two- or three-dimensions. Flow cytometry and droplet microfluidics are the two most common methods of flow-through analysis. While both are high-throughputmore » techniques, their difference lays in the fact that the droplet encapsulated cells experience a restricted and personal microenvironment, while in flow cytometry cells experience similar nutrient and stimuli initial concentrations. These methods are rather well established; however, they recently enabled immense strides in single cell phenotypic analysis, namely the identification and analysis of metabolically distinct individuals from an isogenic population using both droplet microfluidics and flow cytometry.« less

Microfluidic integrated acoustic waving for manipulation of cells and molecules.

PubMed

Barani, Alireza; Paktinat, Hossein; Janmaleki, Mohsen; Mohammadi, Aminollah; Mosaddegh, Peiman; Fadaei-Tehrani, Alireza; Sanati-Nezhad, Amir

2016-11-15

Acoustophoresis with its simple and low-cost fabrication, rapid and localized fluid actuation, compatibility with microfluidic components, and biocompatibility for cellular studies, has been extensively integrated into microfluidics to provide on-chip microdevices for a variety of applications in biology, bioengineering and chemistry. Among different applications, noninvasive manipulation of cells and biomolecules are significantly important, which are addressed by acoustic-based microfluidics. Here in this paper, we briefly explain the principles and different configurations of acoustic wave and acoustic streaming for the manipulation of cells and molecules and overview its applications for single cell isolation, cell focusing and sorting, cell washing and patterning, cell-cell fusion and communication, and tissue engineering. We further discuss the application of acoustic-based microfluidic systems for the mixing and transport of liquids, manipulation of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules, followed by explanation on the present challenges of acoustic-based microfluidics for the handling of cells and molecules, and highlighting the future directions. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Directed Assembly of Cells with Magnetic Nanowires

NASA Astrophysics Data System (ADS)

Tanase, M.; Hultgren, A.; Chen, C. S.; Reich, D. H.

2003-03-01

We demonstrate the use of magnetic nanowires for assembly and manipulation of mammalian cells. Currently, superparamagnetic beads are used for manipulations of cells, but large field strengths and gradients are required for these to be effective. Unlike the beads, the large remnant magnetization of the nanowires offers the prospect of a variety of low-field manipulation techniques. Ferromagnetic nanowires suspended in fluids can be easily manipulated and assembled using small magnetic field [1]. The wires can be bound to cells, and the dipolar interaction between the nanowires can be used to create self-assembled cell chains. Microfabricated arrays of Py magnets were used to trap single cells or chains of cells bound to Ni nanowires. Possible applications of these techniques include controlled initiation of cell cultures, as well as isolation of individual cells. This work was supported by DARPA/AFOSR Grant No. F49620-02-1-0307 and by the David and Lucile Packard Foundation Grant No. 2001-17715. [1] M. Tanase et.al., Nanoletters 1, 155 (2001), J. Appl. Phys. 91, 8549 (2002).

Ultrahigh Frequency Lensless Ultrasonic Transducers for Acoustic Tweezers Application

PubMed Central

Hsu, Hsiu-Sheng; Li, Ying; Lee, Changyang; Lin, Anderson; Zhou, Qifa; Kim, Eun Sok; Shung, Kirk Koping

2014-01-01

Similar to optical tweezers, a tightly focused ultrasound microbeam is needed to manipulate microparticles in acoustic tweezers. The development of highly sensitive ultrahigh frequency ultrasonic transducers is crucial for trapping particles or cells with a size of a few microns. As an extra lens would cause excessive attenuation at ultrahigh frequencies, two types of 200-MHz lensless transducer design were developed as an ultrasound microbeam device for acoustic tweezers application. Lithium niobate single crystal press-focused (PF) transducer and zinc oxide self-focused transducer were designed, fabricated and characterized. Tightly focused acoustic beams produced by these transducers were shown to be capable of manipulating single microspheres as small as 5 μm two-dimensionally within a range of hundreds of micrometers in distilled water. The size of the trapped microspheres is the smallest ever reported in the literature of acoustic PF devices. These results suggest that these lensless ultrahigh frequency ultrasonic transducers are capable of manipulating particles at the cellular level and that acoustic tweezers may be a useful tool to manipulate a single cell or molecule for a wide range of biomedical applications. PMID:23042219

Hybrid cell adhesive material for instant dielectrophoretic cell trapping and long-term cell function assessment.

PubMed

Reyes, Darwin R; Hong, Jennifer S; Elliott, John T; Gaitan, Michael

2011-08-16

Dielectrophoresis (DEP) for cell manipulation has focused, for the most part, on approaches for separation/enrichment of cells of interest. Advancements in cell positioning and immobilization onto substrates for cell culture, either as single cells or as cell aggregates, has benefited from the intensified research efforts in DEP (electrokinetic) manipulation. However, there has yet to be a DEP approach that provides the conditions for cell manipulation while promoting cell function processes such as cell differentiation. Here we present the first demonstration of a system that combines DEP with a hybrid cell adhesive material (hCAM) to allow for cell entrapment and cell function, as demonstrated by cell differentiation into neuronlike cells (NLCs). The hCAM, comprised of polyelectrolytes and fibronectin, was engineered to function as an instantaneous cell adhesive surface after DEP manipulation and to support long-term cell function (cell proliferation, induction, and differentiation). Pluripotent P19 mouse embryonal carcinoma cells flowing within a microchannel were attracted to the DEP electrode surface and remained adhered onto the hCAM coating under a fluid flow field after the DEP forces were removed. Cells remained viable after DEP manipulation for up to 8 d, during which time the P19 cells were induced to differentiate into NLCs. This approach could have further applications in areas such as cell-cell communication, three-dimensional cell aggregates to create cell microenvironments, and cell cocultures.

Exploring protein-DNA interactions in 3D using in situ construction, manipulation, and visualization of individual DNA-dumbbells with optical traps, microfluidics, and fluorescence microscopy

PubMed Central

Forget, Anthony L.; Dombrowski, Christopher C.; Amitani, Ichiro; Kowalczykowski, Stephen C.

2015-01-01

In this Protocol, we describe a procedure to generate ‘DNA-dumbbells’ — single molecules of DNA with a microscopic bead attached at each end — and techniques for manipulating individual DNA-dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA-dumbbells and to visualize individual protein–DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free ‘reservoir’. The reservoir provides the means to examine formation of DNA–protein complexes in solution in the absence of external flow forces, while still maintaining a predetermined end-to-end extension of the DNA. These features facilitate examination of the role of three-dimensional DNA conformation and dynamics in protein–DNA interactions. Preparation of flow cells and reagents requires two days each; in situ DNA-dumbbell assembly and imaging of single protein–DNA complexes requires another day. PMID:23411634

Transportation of single cell and microbubbles by phase-shift introduced to standing leaky surface acoustic waves

PubMed Central

Meng, Long; Cai, Feiyan; Zhang, Zidong; Niu, Lili; Jin, Qiaofeng; Yan, Fei; Wu, Junru; Wang, Zhanhui; Zheng, Hairong

2011-01-01

A microfluidic device was developed to precisely transport a single cell or multiple microbubbles by introducing phase-shifts to a standing leaky surface acoustic wave (SLSAW). The device consists of a polydimethyl-siloxane (PDMS) microchannel and two phase-tunable interdigital transducers (IDTs) for the generation of the relative phase for the pair of surface acoustic waves (SAW) propagating along the opposite directions forming a standing wave. When the SAW contacts the fluid medium inside the microchannel, some of SAW energy is coupled to the fluid and the SAW becomes the leaky surface wave. By modulating the relative phase between two IDTs, the positions of pressure nodes of the SLSAW in the microchannel change linearly resulting in the transportation of a single cell or microbubbles. The results also reveal that there is a good linear relationship between the relative phase and the displacement of a single cell or microbubbles. Furthermore, the single cell and the microbubbles can be transported over a predetermined distance continuously until they reach the targeted locations. This technique has its distinct advantages, such as precise position-manipulation, simple to implement, miniature size, and noninvasive character, which may provide an effective method for the position-manipulation of a single cell and microbubbles in many biological and biomedical applications. PMID:22662056

Simultaneous cellular-resolution optical perturbation and imaging of place cell firing fields

PubMed Central

Rickgauer, John Peter; Deisseroth, Karl; Tank, David W.

2015-01-01

Linking neural microcircuit function to emergent properties of the mammalian brain requires fine-scale manipulation and measurement of neural activity during behavior, where each neuron’s coding and dynamics can be characterized. We developed an optical method for simultaneous cellular-resolution stimulation and large-scale recording of neuronal activity in behaving mice. Dual-wavelength two-photon excitation allowed largely independent functional imaging with a green fluorescent calcium sensor (GCaMP3, λ = 920 ± 6 nm) and single-neuron photostimulation with a red-shifted optogenetic probe (C1V1, λ = 1,064 ± 6 nm) in neurons coexpressing the two proteins. We manipulated task-modulated activity in individual hippocampal CA1 place cells during spatial navigation in a virtual reality environment, mimicking natural place-field activity, or ‘biasing’, to reveal subthreshold dynamics. Notably, manipulating single place-cell activity also affected activity in small groups of other place cells that were active around the same time in the task, suggesting a functional role for local place cell interactions in shaping firing fields. PMID:25402854

Innovative Tools and Technology for Analysis of Single Cells and Cell-Cell Interaction.

PubMed

Konry, Tania; Sarkar, Saheli; Sabhachandani, Pooja; Cohen, Noa

2016-07-11

Heterogeneity in single-cell responses and intercellular interactions results from complex regulation of cell-intrinsic and environmental factors. Single-cell analysis allows not only detection of individual cellular characteristics but also correlation of genetic content with phenotypic traits in the same cell. Technological advances in micro- and nanofabrication have benefited single-cell analysis by allowing precise control of the localized microenvironment, cell manipulation, and sensitive detection capabilities. Additionally, microscale techniques permit rapid, high-throughput, multiparametric screening that has become essential for -omics research. This review highlights innovative applications of microscale platforms in genetic, proteomic, and metabolic detection in single cells; cell sorting strategies; and heterotypic cell-cell interaction. We discuss key design aspects of single-cell localization and isolation in microfluidic systems, dynamic and endpoint analyses, and approaches that integrate highly multiplexed detection of various intracellular species.

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

NASA Astrophysics Data System (ADS)

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.

Automated single cell sorting and deposition in submicroliter drops

NASA Astrophysics Data System (ADS)

Salánki, Rita; Gerecsei, Tamás; Orgovan, Norbert; Sándor, Noémi; Péter, Beatrix; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

2014-08-01

Automated manipulation and sorting of single cells are challenging, when intact cells are needed for further investigations, e.g., RNA or DNA sequencing. We applied a computer controlled micropipette on a microscope admitting 80 PCR (Polymerase Chain Reaction) tubes to be filled with single cells in a cycle. Due to the Laplace pressure, fluid starts to flow out from the micropipette only above a critical pressure preventing the precise control of drop volume in the submicroliter range. We found an anomalous pressure additive to the Laplace pressure that we attribute to the evaporation of the drop. We have overcome the problem of the critical dropping pressure with sequentially operated fast fluidic valves timed with a millisecond precision. Minimum drop volume was 0.4-0.7 μl with a sorting speed of 15-20 s per cell. After picking NE-4C neuroectodermal mouse stem cells and human primary monocytes from a standard plastic Petri dish we could gently deposit single cells inside tiny drops. 94 ± 3% and 54 ± 7% of the deposited drops contained single cells for NE-4C and monocytes, respectively. 7.5 ± 4% of the drops contained multiple cells in case of monocytes. Remaining drops were empty. Number of cells deposited in a drop could be documented by imaging the Petri dish before and after sorting. We tuned the adhesion force of cells to make the manipulation successful without the application of microstructures for trapping cells on the surface. We propose that our straightforward and flexible setup opens an avenue for single cell isolation, critically needed for the rapidly growing field of single cell biology.

High accuracy indirect optical manipulation of live cells with functionalized microtools

NASA Astrophysics Data System (ADS)

Vizsnyiczai, Gaszton; Aekbote, Badri L.; Buzás, András.; Grexa, István.; Ormos, Pál.; Kelemen, Lóránd

2016-09-01

Optical micro manipulation of live cells has been extensively used to study a wide range of cellular phenomena with relevance in basic research or in diagnostics. The approaches span from manipulation of many cells for high throughput measurement or sorting, to more elaborated studies of intracellular events on trapped single cells when coupled with modern imaging techniques. In case of direct cell trapping the damaging effects of light-cell interaction must be minimized, for instance with the choice of proper laser wavelength. Microbeads have already been used for trapping cells indirectly thereby reducing the irradiation damage and increasing trapping efficiency with their high refractive index contrast. We show here that such intermediate objects can be tailor-made for indirect cell trapping to further increase cell-to-focal spot distance while maintaining their free and fast maneuverability. Carefully designed structures were produced with two-photon polymerization with shapes optimized for effective manipulation and cell attachment. Functionalization of the microstructures is also presented that enables cell attachment to them within a few seconds with strength much higher that the optical forces. Fast cell actuation in 6 degrees of freedom is demonstrated with the outlook to possible applications in cell imaging.

Single-Cell Microgels: Technology, Challenges, and Applications.

PubMed

Kamperman, Tom; Karperien, Marcel; Le Gac, Séverine; Leijten, Jeroen

2018-04-12

Single-cell-laden microgels effectively act as the engineered counterpart of the smallest living building block of life: a cell within its pericellular matrix. Recent breakthroughs have enabled the encapsulation of single cells in sub-100-μm microgels to provide physiologically relevant microniches with minimal mass transport limitations and favorable pharmacokinetic properties. Single-cell-laden microgels offer additional unprecedented advantages, including facile manipulation, culture, and analysis of individual cell within 3D microenvironments. Therefore, single-cell microgel technology is expected to be instrumental in many life science applications, including pharmacological screenings, regenerative medicine, and fundamental biological research. In this review, we discuss the latest trends, technical challenges, and breakthroughs, and present our vision of the future of single-cell microgel technology and its applications. Copyright © 2018 Elsevier Ltd. All rights reserved.

The future is now: single-cell genomics of bacteria and archaea

PubMed Central

Blainey, Paul C.

2013-01-01

Interest in the expanding catalog of uncultivated microorganisms, increasing recognition of heterogeneity among seemingly similar cells, and technological advances in whole-genome amplification and single-cell manipulation are driving considerable progress in single-cell genomics. Here, the spectrum of applications for single-cell genomics, key advances in the development of the field, and emerging methodology for single-cell genome sequencing are reviewed by example with attention to the diversity of approaches and their unique characteristics. Experimental strategies transcending specific methodologies are identified and organized as a road map for future studies in single-cell genomics of environmental microorganisms. Over the next decade, increasingly powerful tools for single-cell genome sequencing and analysis will play key roles in accessing the genomes of uncultivated organisms, determining the basis of microbial community functions, and fundamental aspects of microbial population biology. PMID:23298390

Hydrodynamic lift for single cell manipulation in a femtosecond laser fabricated optofluidic chip

NASA Astrophysics Data System (ADS)

Bragheri, Francesca; Osellame, Roberto

2017-08-01

Single cell sorting based either on fluorescence or on mechanical properties has been exploited in the last years in microfluidic devices. Hydrodynamic focusing allows increasing the efficiency of theses devices by improving the matching between the region of optical analysis and that of cell flow. Here we present a very simple solution fabricated by femtosecond laser micromachining that exploits flow laminarity in microfluidic channels to easily lift the sample flowing position to the channel portion illuminated by the optical waveguides used for single cell trapping and analysis.

Report of the Microbial Development Working Group

NASA Technical Reports Server (NTRS)

Nelson, G.

1985-01-01

In formulating ideas on the relationship of gravity to the development, growth, and reproduction of microorganisms, a rather liberal definition of microorganisms is used which includes bacteria, yeasts, protists, filamentous fungi, and single cells in culture. A principal advantage of microorganisms as experimental subjects is the rigor with which they can be defined and controlled. As single cells, each cell may be regarded as identical to the others in the population. This property applies to the morphology, physiology, and genetic parameters of the cells. The growth and development of the population is subject to precise manipulation as the nutritional requirements are known and minimal media formulations have been developed. Growth and differentiation can be manipulated in a variety of ways, such as alteration of the culture temperature and food supply, or by use of mutants. Finally, the short generation times of microorganisms provide the opportunity to conduct multigenerational studies within practical time limits and, in a similar vein, cellular responses to various stimuli or stresses are conveniently monitored because of the rapid response times of single cells.

Single-neuron labeling with inducible cre-mediated knockout in transgenic mice

PubMed Central

Young, Paul; Qiu, Li; Wang, Dongqing; Zhao, Shengli; Gross, James; Feng, Guoping

2011-01-01

To facilitate functional analysis of neuronal connectivity in a mammalian nervous system tightly packed with billions of cells, we developed a new technique that allows inducible genetic manipulations within fluorescently labeled single neurons in mice. We term this technique SLICK for Single-neuron Labeling with Inducible Cre-mediated Knockout. SLICK is achieved by co-expressing a drug-inducible form of cre recombinase and a fluorescent protein within the same small subsets of neurons. Thus, SLICK combines the powerful cre recombinase system for conditional genetic manipulation and the fluorescent labeling of single neurons for imaging. We demonstrate efficient inducible genetic manipulation in several types of neurons using SLICK. Furthermore, we apply SLICK to eliminate synaptic transmission in a small subset of neuromuscular junctions. Our results provide evidence for the long-term stability of inactive neuromuscular synapses in adult animals. More broadly, these studies demonstrate a cre-LoxP compatible system for dissecting gene functions in single identifiable neurons. PMID:18454144

Laser-assisted patch clamping: a methodology

NASA Technical Reports Server (NTRS)

Henriksen, G. H.; Assmann, S. M.; Evans, M. L. (Principal Investigator)

1997-01-01

Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

Raman sorting and identification of single living micro-organisms with optical tweezers

NASA Astrophysics Data System (ADS)

Xie, Changan; Chen, De; Li, Yong-Qing

2005-07-01

We report on a novel technique for sorting and identification of single biological cells and food-borne bacteria based on laser tweezers and Raman spectroscopy (LTRS). With this technique, biological cells of different physiological states in a sample chamber were identified by their Raman spectral signatures and then they were selectively manipulated into a clean collection chamber with optical tweezers through a microchannel. As an example, we sorted the live and dead yeast cells into the collection chamber and validated this with a standard staining technique. We also demonstrated that bacteria existing in spoiled foods could be discriminated from a variety of food particles based on their characteristic Raman spectra and then isolated with laser manipulation. This label-free LTRS sorting technique may find broad applications in microbiology and rapid examination of food-borne diseases.

Isolation of a single rice chromosome by optical micromanipulation

NASA Astrophysics Data System (ADS)

Wang, Haowei; Liu, Xiaohui; Li, Yinmei; Han, Bin; Lou, Liren; Wang, Kangjun

2004-01-01

A new method based on optical tweezers technology is reported for the isolation of a single chromosome. A rice cell suspended in liquid was first fragmented by laser pulses (optical scalpel). Then a single released chromosome from the cell was manipulated and pulled away from other cells and oddments by optical tweezers without any direct mechanical contact. Finally the isolated single chromosome was extracted individually into a glass capillary nearby. After molecular cloning of the isolated chromosome, we obtained some specific DNA segments from the single chromosome. All these segments can be used for rice genomic sequencing. Different methods of extracting a single chromosome are compared. The advantages of optical micromanipulation method are summarized.

Ferromagnetic nanowires: Field-induced self-assembly, magnetotransport and biological applications

NASA Astrophysics Data System (ADS)

Tanase, Monica

In this dissertation, a series of experiments on magnetic nanowires are described. Magnetic nanowires suspended in fluid solutions can be assembled and ordered by taking advantage of their large shape anisotropy. Magnetic manipulation and assembly techniques were developed, using electrodeposited Ni nanowires. Preorienting nanowires in a small magnetic field induced their self-assembly in continuous chains. A new technique of magnetic trapping allowed capture of single nanowires from fluid suspension on lithographically fabricated micromagnets. As described herein, the presence of an external magnetic field plays a fundamental role in all fluid assembly methods used. The dynamics of both chaining and trapping processes is described quantitatively in terms of the interplay of magnetic forces and fluid drag at low Reynolds number. Lithographic methods for addressing single nanowires for transport characterization were developed. Magnetotransport measurements were performed on individual straight and bent PtNiPt nanowires. The Pt end segments provided an oxide-free interface to the magnetic central segment. In straight nanowires, domain reversal was observed to occur via curling mode initiated in a small nucleation volume. Magnetotransport in bent nanowires allowed the investigation of a domain wall trapped at the bend. Magnetic trapping of nanowires on pre-fabricated electrodes was adapted as a successful alternative contacting technique to lithography. The self-assembly and manipulation techniques were adapted for manipulation of cells as nanowires were found to bind to cells through nonspecific adhesion mechanisms. Ni nanowires were found to outperform superparamagnetic beads in magnetic cell separations. Additionally, the large remnant magnetization of the nanowires allowed for low-field manipulation techniques. Self-assembled chains of cells were formed and single cells were localized on substrates patterned with micromagnets. A fluid flow method was developed to controllably introduce the cells in the proximity of arrays of micromagnets. Cells decorated the arrays forming patterns described well by dipolar interactions between the magnetic elements and the nanowires. Calculations of the locations favorable for trapping were performed by evaluating the energy of interaction between the array and the nanowires. A second-order mechanism of cell capture was also identified, i.e. chaining by wire-wire dipolar interaction.

Characterization of the cellular response triggered by gold nanoparticle-mediated laser manipulation

NASA Astrophysics Data System (ADS)

Kalies, Stefan; Keil, Sebastian; Sender, Sina; Hammer, Susanne C.; Antonopoulos, Georgios C.; Schomaker, Markus; Ripken, Tammo; Escobar, Hugo Murua; Meyer, Heiko; Heinemann, Dag

2015-11-01

Laser-based transfection techniques have proven high applicability in several cell biologic applications. The delivery of different molecules using these techniques has been extensively investigated. In particular, new high-throughput approaches such as gold nanoparticle-mediated laser transfection allow efficient delivery of antisense molecules or proteins into cells preserving high cell viabilities. However, the cellular response to the perforation procedure is not well understood. We herein analyzed the perforation kinetics of single cells during resonant gold nanoparticle-mediated laser manipulation with an 850-ps laser system at a wavelength of 532 nm. Inflow velocity of propidium iodide into manipulated cells reached a maximum within a few seconds. Experiments based on the inflow of FM4-64 indicated that the membrane remains permeable for a few minutes for small molecules. To further characterize the cellular response postmanipulation, we analyzed levels of oxidative heat or general stress. Although we observed an increased formation of reactive oxygen species by an increase of dichlorofluorescein fluorescence, heat shock protein 70 was not upregulated in laser-treated cells. Additionally, no evidence of stress granule formation was visible by immunofluorescence staining. The data provided in this study help to identify the cellular reactions to gold nanoparticle-mediated laser manipulation.

Dependence of Impedance of Embedded Single Cells on Cellular Behaviour.

PubMed

Cho, Sungbo; Castellarnau, Marc; Samitier, Josep; Thielecke, Hagen

2008-02-21

Non-invasive single cell analyses are increasingly required for the medicaldiagnostics of test substances or the development of drugs and therapies on the single celllevel. For the non-invasive characterisation of cells, impedance spectroscopy whichprovides the frequency dependent electrical properties has been used. Recently,microfludic systems have been investigated to manipulate the single cells and tocharacterise the electrical properties of embedded cells. In this article, the impedance ofpartially embedded single cells dependent on the cellular behaviour was investigated byusing the microcapillary. An analytical equation was derived to relate the impedance ofembedded cells with respect to the morphological and physiological change ofextracellular interface. The capillary system with impedance measurement showed afeasibility to monitor the impedance change of embedded single cells caused bymorphological and physiological change of cell during the addition of DMSO. By fittingthe derived equation to the measured impedance of cell embedded at different negativepressure levels, it was able to extrapolate the equivalent gap and gap conductivity betweenthe cell and capillary wall representing the cellular behaviour.

Single Vector Calibration System for Multi-Axis Load Cells and Method for Calibrating a Multi-Axis Load Cell

NASA Technical Reports Server (NTRS)

Parker, Peter A. (Inventor)

2003-01-01

A single vector calibration system is provided which facilitates the calibration of multi-axis load cells, including wind tunnel force balances. The single vector system provides the capability to calibrate a multi-axis load cell using a single directional load, for example loading solely in the gravitational direction. The system manipulates the load cell in three-dimensional space, while keeping the uni-directional calibration load aligned. The use of a single vector calibration load reduces the set-up time for the multi-axis load combinations needed to generate a complete calibration mathematical model. The system also reduces load application inaccuracies caused by the conventional requirement to generate multiple force vectors. The simplicity of the system reduces calibration time and cost, while simultaneously increasing calibration accuracy.

Revealing Abrupt and Spontaneous Ruptures of Protein Native Structure under picoNewton Compressive Force Manipulation.

PubMed

Chowdhury, S Roy; Cao, Jin; He, Yufan; Lu, H Peter

2018-03-27

Manipulating protein conformations for exploring protein structure-function relationship has shown great promise. Although protein conformational changes under pulling force manipulation have been extensively studied, protein conformation changes under a compressive force have not been explored quantitatively. The latter is even more biologically significant and relevant in revealing protein functions in living cells associated with protein crowdedness, distribution fluctuations, and cell osmotic stress. Here we report our experimental observations on abrupt ruptures of protein native structures under compressive force, demonstrated and studied by single-molecule AFM-FRET spectroscopic nanoscopy. Our results show that the protein ruptures are abrupt and spontaneous events occurred when the compressive force reaches a threshold of 12-75 pN, a force amplitude accessible from thermal fluctuations in a living cell. The abrupt ruptures are sensitive to local environment, likely a general and important pathway of protein unfolding in living cells.

System-Level Biochip for Impedance Sensing and Programmable Manipulation of Bladder Cancer Cells

PubMed Central

Chuang, Cheng-Hsin; Huang, Yao-Wei; Wu, Yao-Tung

2011-01-01

This paper develops a dielectrophoretic (DEP) chip with multi-layer electrodes and a micro-cavity array for programmable manipulations of cells and impedance measurement. The DEP chip consists of an ITO top electrode, flow chamber, middle electrode on an SU-8 surface, micro-cavity arrays of SU-8 and distributed electrodes at the bottom of the micro-cavity. Impedance sensing of single cells could be performed as follows: firstly, cells were trapped in a micro-cavity array by negative DEP force provided by top and middle electrodes; then, the impedance measurement for discrimination of different stage of bladder cancer cells was accomplished by the middle and bottom electrodes. After impedance sensing, the individual releasing of trapped cells was achieved by negative DEP force using the top and bottom electrodes in order to collect the identified cells once more. Both cell manipulations and impedance measurement had been integrated within a system controlled by a PC-based LabVIEW program. In the experiments, two different stages of bladder cancer cell lines (grade III: T24 and grade II: TSGH8301) were utilized for the demonstration of programmable manipulation and impedance sensing; as the results show, the lower-grade bladder cancer cells (TSGH8301) possess higher impedance than the higher-grade ones (T24). In general, the multi-step manipulations of cells can be easily programmed by controlling the electrical signal in our design, which provides an excellent platform technology for lab-on-a-chip (LOC) or a micro-total-analysis-system (Micro TAS). PMID:22346685

Cloning of Plasmodium falciparum by single-cell sorting

PubMed Central

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

Evidence of femtosecond-laser pulse induced cell membrane nanosurgery

NASA Astrophysics Data System (ADS)

Katchinskiy, Nir; Godbout, Roseline; Elezzabi, Abdulhakem Y.

2017-02-01

The mechanism of femtosecond laser nanosurgical attachment is investigated in the following article. Using sub-10 femtosecond laser pulses with 800 nm central wavelength were used to attach retinoblastoma cells. During the attachment process the cell membrane phospholipid bilayers hemifuse into one shared phospholipid bilayer, at the location of attachment. Transmission electron microscopy was used in order to verify the above hypothesis. Based on the imaging results, it was concluded that the two cell membrane coalesce to form one single shared membrane. The technique of cell-cell attachment via femtosecond laser pulses could potentially serve as a platform for precise cell membrane manipulation. Manipulation of the cellular membrane is valuable for studying diseases such as cancer; where the expression level of plasma proteins on the cell membrane is altered.

Holographic acoustic elements for manipulation of levitated objects.

PubMed

Marzo, Asier; Seah, Sue Ann; Drinkwater, Bruce W; Sahoo, Deepak Ranjan; Long, Benjamin; Subramanian, Sriram

2015-10-27

Sound can levitate objects of different sizes and materials through air, water and tissue. This allows us to manipulate cells, liquids, compounds or living things without touching or contaminating them. However, acoustic levitation has required the targets to be enclosed with acoustic elements or had limited manoeuvrability. Here we optimize the phases used to drive an ultrasonic phased array and show that acoustic levitation can be employed to translate, rotate and manipulate particles using even a single-sided emitter. Furthermore, we introduce the holographic acoustic elements framework that permits the rapid generation of traps and provides a bridge between optical and acoustical trapping. Acoustic structures shaped as tweezers, twisters or bottles emerge as the optimum mechanisms for tractor beams or containerless transportation. Single-beam levitation could manipulate particles inside our body for applications in targeted drug delivery or acoustically controlled micro-machines that do not interfere with magnetic resonance imaging.

Holographic acoustic elements for manipulation of levitated objects

NASA Astrophysics Data System (ADS)

Marzo, Asier; Seah, Sue Ann; Drinkwater, Bruce W.; Sahoo, Deepak Ranjan; Long, Benjamin; Subramanian, Sriram

2015-10-01

Sound can levitate objects of different sizes and materials through air, water and tissue. This allows us to manipulate cells, liquids, compounds or living things without touching or contaminating them. However, acoustic levitation has required the targets to be enclosed with acoustic elements or had limited manoeuvrability. Here we optimize the phases used to drive an ultrasonic phased array and show that acoustic levitation can be employed to translate, rotate and manipulate particles using even a single-sided emitter. Furthermore, we introduce the holographic acoustic elements framework that permits the rapid generation of traps and provides a bridge between optical and acoustical trapping. Acoustic structures shaped as tweezers, twisters or bottles emerge as the optimum mechanisms for tractor beams or containerless transportation. Single-beam levitation could manipulate particles inside our body for applications in targeted drug delivery or acoustically controlled micro-machines that do not interfere with magnetic resonance imaging.

Holographic acoustic elements for manipulation of levitated objects

PubMed Central

Marzo, Asier; Seah, Sue Ann; Drinkwater, Bruce W.; Sahoo, Deepak Ranjan; Long, Benjamin; Subramanian, Sriram

2015-01-01

Sound can levitate objects of different sizes and materials through air, water and tissue. This allows us to manipulate cells, liquids, compounds or living things without touching or contaminating them. However, acoustic levitation has required the targets to be enclosed with acoustic elements or had limited manoeuvrability. Here we optimize the phases used to drive an ultrasonic phased array and show that acoustic levitation can be employed to translate, rotate and manipulate particles using even a single-sided emitter. Furthermore, we introduce the holographic acoustic elements framework that permits the rapid generation of traps and provides a bridge between optical and acoustical trapping. Acoustic structures shaped as tweezers, twisters or bottles emerge as the optimum mechanisms for tractor beams or containerless transportation. Single-beam levitation could manipulate particles inside our body for applications in targeted drug delivery or acoustically controlled micro-machines that do not interfere with magnetic resonance imaging. PMID:26505138

Laser-mediated perforation of plant cells

NASA Astrophysics Data System (ADS)

Wehner, Martin; Jacobs, Philipp; Esser, Dominik; Schinkel, Helga; Schillberg, Stefan

2007-07-01

The functional analysis of plant cells at the cellular and subcellular levels requires novel technologies for the directed manipulation of individual cells. Lasers are increasingly exploited for the manipulation of plant cells, enabling the study of biological processes on a subcellular scale including transformation to generate genetically modified plants. In our setup either a picosecond laser operating at 1064 nm wavelength or a continuous wave laser diode emitting at 405 nm are coupled into an inverse microscope. The beams are focused to a spot size of about 1.5 μm and the tobacco cell protoplasts are irradiated. Optoporation is achieved when targeting the laser focal spot at the outermost edge of the plasma membrane. In case of the picosecond laser a single pulse with energy of about 0.4 μJ was sufficient to perforate the plasma membrane enabling the uptake of dye or DNA from the surrounding medium into the cytosol. When the ultraviolet laser diode at a power level of 17 mW is employed an irradiation time of 200 - 500 milliseconds is necessary to enable the uptake of macromolecules. In the presence of an EYFP encoding plasmid with a C-terminal peroxisomal signal sequence in the surrounding medium transient transformation of tobacco protoplasts could be achieved in up to 2% of the optoporated cells. Single cell perforation using this novel optoporation method shows that isolated plant cells can be permeabilized without direct manipulation. This is a valuable procedure for cell-specific applications, particularly where the import of specific molecules into plant cells is required for functional analysis.

Manipulation of acoustic wavefront by gradient metasurface based on Helmholtz Resonators.

PubMed

Lan, Jun; Li, Yifeng; Xu, Yue; Liu, Xiaozhou

2017-09-06

We designed a gradient acoustic metasurface to manipulate acoustic wavefront freely. The broad bandwidth and high efficiency transmission are achieved by the acoustic metasurface which is constructed with a series of unit cells to provide desired discrete acoustic velocity distribution. Each unit cell is composed of a decorated metal plate with four periodically arrayed Helmholtz resonators (HRs) and a single slit. The design employs a gradient velocity to redirect refracted wave and the impedance matching between the metasurface and the background medium can be realized by adjusting the slit width of unit cell. The theoretical and numerical results show that some excellent wavefront manipulations are demonstrated by anomalous refraction, non-diffracting Bessel beam, sub-wavelength flat focusing, and effective tunable acoustic negative refraction. Our designed structure may offer potential applications for the imaging system, beam steering and acoustic lens.

Optical trapping for analytical biotechnology.

PubMed

Ashok, Praveen C; Dholakia, Kishan

2012-02-01

We describe the exciting advances of using optical trapping in the field of analytical biotechnology. This technique has opened up opportunities to manipulate biological particles at the single cell or even at subcellular levels which has allowed an insight into the physical and chemical mechanisms of many biological processes. The ability of this technique to manipulate microparticles and measure pico-Newton forces has found several applications such as understanding the dynamics of biological macromolecules, cell-cell interactions and the micro-rheology of both cells and fluids. Furthermore we may probe and analyse the biological world when combining trapping with analytical techniques such as Raman spectroscopy and imaging. Copyright © 2011 Elsevier Ltd. All rights reserved.

Optical Manipulation of Single Magnetic Beads in a Microwell Array on a Digital Microfluidic Chip.

PubMed

Decrop, Deborah; Brans, Toon; Gijsenbergh, Pieter; Lu, Jiadi; Spasic, Dragana; Kokalj, Tadej; Beunis, Filip; Goos, Peter; Puers, Robert; Lammertyn, Jeroen

2016-09-06

The detection of single molecules in magnetic microbead microwell array formats revolutionized the development of digital bioassays. However, retrieval of individual magnetic beads from these arrays has not been realized until now despite having great potential for studying captured targets at the individual level. In this paper, optical tweezers were implemented on a digital microfluidic platform for accurate manipulation of single magnetic beads seeded in a microwell array. Successful optical trapping of magnetic beads was found to be dependent on Brownian motion of the beads, suggesting a 99% chance of trapping a vibrating bead. A tailor-made experimental design was used to screen the effect of bead type, ionic buffer strength, surfactant type, and concentration on the Brownian activity of beads in microwells. With the optimal conditions, the manipulation of magnetic beads was demonstrated by their trapping, retrieving, transporting, and repositioning to a desired microwell on the array. The presented platform combines the strengths of digital microfluidics, digital bioassays, and optical tweezers, resulting in a powerful dynamic microwell array system for single molecule and single cell studies.

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells

PubMed Central

Cole, Russell H.; Tang, Shi-Yang; Siltanen, Christian A.; Shahi, Payam; Zhang, Jesse Q.; Poust, Sean; Gartner, Zev J.; Abate, Adam R.

2017-01-01

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay. PMID:28760972

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells.

PubMed

Cole, Russell H; Tang, Shi-Yang; Siltanen, Christian A; Shahi, Payam; Zhang, Jesse Q; Poust, Sean; Gartner, Zev J; Abate, Adam R

2017-08-15

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

Printed droplet microfluidics for on demand dispensing of picoliter droplets and cells

NASA Astrophysics Data System (ADS)

Cole, Russell H.; Tang, Shi-Yang; Siltanen, Christian A.; Shahi, Payam; Zhang, Jesse Q.; Poust, Sean; Gartner, Zev J.; Abate, Adam R.

2017-08-01

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

Living cell manipulation, manageable sampling, and shotgun picoliter electrospray mass spectrometry for profiling metabolites.

PubMed

Gholipour, Yousef; Erra-Balsells, Rosa; Hiraoka, Kenzo; Nonami, Hiroshi

2013-02-01

A modified cell pressure probe and an online Orbitrap mass spectrometer were used to sample in situ plant single cells without any additional manipulation. The cell pressure probe, a quartz capillary tip filled with an oil mixture, was penetrated to various depths into parenchyma cells of tulip bulb scale, followed by a hydraulic continuity test to determine the exact location of the tip inside target cells. The operation was conducted under a digital microscope, and the capillary tip was photographed to calculate the volume of the cell sap sucked. The cell sap sample was then directly nebulized/ionized under high-voltage conditions at the entrance of the mass spectrometer. Several sugars, amino acids, organic acids, vitamins, fatty acids, and secondary metabolites were detected. Because picoliter solutions can be accurately handled and measured, known volumes of standard solutions can be added to cell sap samples inside the capillary tip to be used as references for metabolite characterization and relative quantitation. The high precision and sensitivity of the cell pressure probe and Orbitrap mass spectrometer allow for the manipulation and analysis of both femtoliter cell sap samples and standard solutions. Copyright © 2012 Elsevier Inc. All rights reserved.

Dependence of Impedance of Embedded Single Cells on Cellular Behaviour

PubMed Central

Cho, Sungbo; Castellarnau, Marc; Samitier, Josep; Thielecke, Hagen

2008-01-01

Non-invasive single cell analyses are increasingly required for the medical diagnostics of test substances or the development of drugs and therapies on the single cell level. For the non-invasive characterisation of cells, impedance spectroscopy which provides the frequency dependent electrical properties has been used. Recently, microfludic systems have been investigated to manipulate the single cells and to characterise the electrical properties of embedded cells. In this article, the impedance of partially embedded single cells dependent on the cellular behaviour was investigated by using the microcapillary. An analytical equation was derived to relate the impedance of embedded cells with respect to the morphological and physiological change of extracellular interface. The capillary system with impedance measurement showed a feasibility to monitor the impedance change of embedded single cells caused by morphological and physiological change of cell during the addition of DMSO. By fitting the derived equation to the measured impedance of cell embedded at different negative pressure levels, it was able to extrapolate the equivalent gap and gap conductivity between the cell and capillary wall representing the cellular behaviour. PMID:27879760

A single-cell and feeder-free culture system for monkey embryonic stem cells.

PubMed

Ono, Takashi; Suzuki, Yutaka; Kato, Yosuke; Fujita, Risako; Araki, Toshihiro; Yamashita, Tomoko; Kato, Hidemasa; Torii, Ryuzo; Sato, Naoya

2014-01-01

Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.

A Minimally Invasive Method for Retrieving Single Adherent Cells of Different Types from Cultures

PubMed Central

Zeng, Jia; Mohammadreza, Aida; Gao, Weimin; Merza, Saeed; Smith, Dean; Kelbauskas, Laimonas; Meldrum, Deirdre R.

2014-01-01

The field of single-cell analysis has gained a significant momentum over the last decade. Separation and isolation of individual cells is an indispensable step in almost all currently available single-cell analysis technologies. However, stress levels introduced by such manipulations remain largely unstudied. We present a method for minimally invasive retrieval of selected individual adherent cells of different types from cell cultures. The method is based on a combination of mechanical (shear flow) force and biochemical (trypsin digestion) treatment. We quantified alterations in the transcription levels of stress response genes in individual cells exposed to varying levels of shear flow and trypsinization. We report optimal temperature, RNA preservation reagents, shear force and trypsinization conditions necessary to minimize changes in the stress-related gene expression levels. The method and experimental findings are broadly applicable and can be used by a broad research community working in the field of single cell analysis. PMID:24957932

Laser microtreatment for genetic manipulations and DNA diagnostics by a combination of microbeam and photonic tweezers (laser microbeam trap)

NASA Astrophysics Data System (ADS)

Greulich, Karl-Otto; Monajembashi, Shamci; Celeda, D.; Endlich, N.; Eickhoff, Holger; Hoyer, Carsten; Leitz, G.; Weber, Gerd; Scheef, J.; Rueterjans, H.

1994-12-01

Genomes of higher organisms are larger than one typically expects. For example, the DNA of a single human cell is almost two meters long, the DNA in the human body covers the distance Earth-Sun approximately 140 times. This is often not considered in typical molecular biological approaches for DNA diagnostics, where usually only DNA of the length of a gene is investigated. Also, one basic aspect of sequencing the human genome is not really solved: the problem how to prepare the huge amounts of DNA required. Approaches from biomedical optics combined with new developments in single molecule biotechnology may at least contribute some parts of the puzzle. A large genome can be partitioned into portions comprising approximately 1% of the whole DNA using a laser microbeam. The single DNA fragment can be amplified by the polymerase chain reaction in order to obtain a sufficient amount of molecules for conventional DNA diagnostics or for analysis by octanucleotide hybridization. When not amplified by biotechnological processes, the individual DNA molecule can be visualized in the light microscope and can be manipulated and dissected with the laser microbeam trap. The DNA probes obtained by single molecule biotechnology can be employed for fluorescence in situ introduced into plant cells and subcellular structures even when other techniques fail. Since the laser microbeam trap allows to work in the interior of a cell without opening it, subcellular structures can be manipulated. For example, in algae, such structures can be moved out of their original position and used to study intracellular viscosities.

Light Activated Cell Migration in Synthetic Extracellular Matrices

PubMed Central

Guo, Qiongyu; Wang, Xiaobo; Tibbitt, Mark W.; Anseth, Kristi S.; Montell, Denise J.; Elisseeff, Jennifer H.

2012-01-01

Synthetic extracellular matrices provide a framework in which cells can be exposed to defined physical and biological cues. However no method exists to manipulate single cells within these matrices. It is desirable to develop such methods in order to understand fundamental principles of cell migration and define conditions that support or inhibit cell movement within these matrices. Here, we present a strategy for manipulating individual mammalian stem cells in defined synthetic hydrogels through selective optical activation of Rac, which is an intracellular signaling protein that plays a key role in cell migration. Photoactivated cell migration in synthetic hydrogels depended on mechanical and biological cues in the biomaterial. Real-time hydrogel photodegradation was employed to create geometrically defined channels and spaces in which cells could be photoactivated to migrate. Cell migration speed was significantly higher in the photo-etched channels and cells could easily change direction of movement compared to the bulk hydrogels. PMID:22889487

Phenotype classification of single cells using SRS microscopy, RNA sequencing, and microfluidics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Streets, Aaron M.; Cao, Chen; Zhang, Xiannian; Huang, Yanyi

2016-03-01

Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.

Lab on a chip for multiplexed immunoassays to detect bladder cancer using multifunctional dielectrophoretic manipulations.

PubMed

Chuang, Cheng-Hsin; Wu, Ting-Feng; Chen, Cheng-Ho; Chang, Kai-Chieh; Ju, Jing-Wei; Huang, Yao-Wei; Van Nhan, Vo

2015-07-21

A multiplexed immunosensor has been developed for the detection of specific biomarkers Galectin-1 (Gal-1) and Lactate Dehydrogenase B (LDH-B) present in different grades of bladder cancer cell lysates. In order to immobilize nanoprobes with different antibodies on a single chip we employed three-step programmable dielectrophoretic manipulations for focusing, guiding and trapping to enhance the fluorescent response and reduce the interference between the two antibody arrays. The chip consisted of a patterned indium tin oxide (ITO) electrode for sensing and a middle fish bone shaped gold electrode for focusing and guiding. Using ITO electrodes for the sensing area can effectively eliminate the background noise of fluorescence response as compared to metal electrodes. It was also observed that the three step manipulation increased fluorescence response after immunosensing by about 4.6 times as compared to utilizing DEP for just trapping the nanoprobes. Two different-grade bladder cancer cell lysates (grade I: RT4 and grade III: T24) were individually analyzed for detecting the protein expression levels of Gal-1 and LDH-B. The fluorescence intensity observed for Gal-1 is higher than that of LDH-B in the T24 cell lysate; however the response observed in RT4 is higher for LDH-B as compared to Gal-1. Thus we can effectively identify the different grades of bladder cancer cells. In addition, the platform for DEP manipulation developed in this study can enable real time detection of multiple analytes on a single chip and provide more practical benefits for clinical diagnosis.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Iwata, Futoshi, E-mail: iwata.futoshi@shizuoka.ac.jp; Research Institute of Electronics, Shizuoka University, Johoku, Naka-ku, Hamamatsu 432-8011; Adachi, Makoto

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

Droplet microfluidics--a tool for single-cell analysis.

PubMed

Joensson, Haakan N; Andersson Svahn, Helene

2012-12-03

Droplet microfluidics allows the isolation of single cells and reagents in monodisperse picoliter liquid capsules and manipulations at a throughput of thousands of droplets per second. These qualities allow many of the challenges in single-cell analysis to be overcome. Monodispersity enables quantitative control of solute concentrations, while encapsulation in droplets provides an isolated compartment for the single cell and its immediate environment. The high throughput allows the processing and analysis of the tens of thousands to millions of cells that must be analyzed to accurately describe a heterogeneous cell population so as to find rare cell types or access sufficient biological space to find hits in a directed evolution experiment. The low volumes of the droplets make very large screens economically viable. This Review gives an overview of the current state of single-cell analysis involving droplet microfluidics and offers examples where droplet microfluidics can further biological understanding. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

Nikitin, V A

2007-01-01

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

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

NASA Astrophysics Data System (ADS)

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.; England, Grant T.; Kim, Philseok; Kolle, Mathias; Ferrante, Thomas; Zarzar, Lauren D.; Strong, Elizabeth; Aizenberg, Joanna

2017-03-01

Mechanical forces in the cell's natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging from the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques.

Nanoliter-Scale Oil-Air-Droplet Chip-Based Single Cell Proteomic Analysis.

PubMed

Li, Zi-Yi; Huang, Min; Wang, Xiu-Kun; Zhu, Ying; Li, Jin-Song; Wong, Catherine C L; Fang, Qun

2018-04-17

Single cell proteomic analysis provides crucial information on cellular heterogeneity in biological systems. Herein, we describe a nanoliter-scale oil-air-droplet (OAD) chip for achieving multistep complex sample pretreatment and injection for single cell proteomic analysis in the shotgun mode. By using miniaturized stationary droplet microreaction and manipulation techniques, our system allows all sample pretreatment and injection procedures to be performed in a nanoliter-scale droplet with minimum sample loss and a high sample injection efficiency (>99%), thus substantially increasing the analytical sensitivity for single cell samples. We applied the present system in the proteomic analysis of 100 ± 10, 50 ± 5, 10, and 1 HeLa cell(s), and protein IDs of 1360, 612, 192, and 51 were identified, respectively. The OAD chip-based system was further applied in single mouse oocyte analysis, with 355 protein IDs identified at the single oocyte level, which demonstrated its special advantages of high enrichment of sequence coverage, hydrophobic proteins, and enzymatic digestion efficiency over the traditional in-tube system.

Cloning of Plasmodium falciparum by single-cell sorting.

PubMed

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.

An open-access microfluidic model for lung-specific functional studies at an air-liquid interface.

PubMed

Nalayanda, Divya D; Puleo, Christopher; Fulton, William B; Sharpe, Leilani M; Wang, Tza-Huei; Abdullah, Fizan

2009-10-01

In an effort to improve the physiologic relevance of existing in vitro models for alveolar cells, we present a microfluidic platform which provides an air-interface in a dynamic system combining microfluidic and suspended membrane culture systems. Such a system provides the ability to manipulate multiple parameters on a single platform along with ease in cell seeding and manipulation. The current study presents a comparison of the efficacy of the hybrid system with conventional platforms using assays analyzing the maintenance of function and integrity of A549 alveolar epithelial cell monolayer cultures. The hybrid system incorporates bio-mimetic nourishment on the basal side of the epithelial cells along with an open system on the apical side of the cells exposed to air allowing for easy access for assays.

Gene editing for cell engineering: trends and applications.

PubMed

Gupta, Sanjeev K; Shukla, Pratyoosh

2017-08-01

Gene editing with all its own advantages in molecular biology applications has made easy manipulation of various production hosts with the discovery and implementation of modern gene editing tools such as Crispr (Clustered regularly interspaced short palindromic repeats), TALENs (Transcription activator-like effector nucleases) and ZFNs (Zinc finger nucleases). With the advent of these modern tools, it is now possible to manipulate the genome of industrial production hosts such as yeast and mammalian cells which allows developing a potential and cost effective recombinant therapeutic protein. These tools also allow single editing to multiple genes for knocking-in or knocking-out of a host genome quickly in an efficient manner. A recent study on "multiplexed" gene editing revolutionized the knock-out and knock-in events of yeast and CHO, mammalian cells genome for metabolic engineering as well as high, stable, and consistent expression of a transgene encoding complex therapeutic protein such as monoclonal antibody. The gene of interest can either be integrated or deleted at single or multiple loci depending on the strategy and production requirement. This review will give a gist of all the modern tools with a brief description and advances in genetic manipulation using three major tools being implemented for the modification of such hosts with the emphasis on the use of Crispr-Cas9 for the "multiplexing gene-editing approach" for genetic manipulation of yeast and CHO mammalian hosts that ultimately leads to a fast track product development with consistent, improved product yield, quality, and thus affordability for a population at large.

Bubble Jet agent release cartridge for chemical single cell stimulation.

PubMed

Wangler, N; Welsche, M; Blazek, M; Blessing, M; Vervliet-Scheebaum, M; Reski, R; Müller, C; Reinecke, H; Steigert, J; Roth, G; Zengerle, R; Paust, N

2013-02-01

We present a new method for the distinct specific chemical stimulation of single cells and small cell clusters within their natural environment. By single-drop release of chemical agents with droplets in size of typical cell diameters (d <30 μm) on-demand micro gradients can be generated for the specific manipulation of single cells. A single channel and a double channel agent release cartridge with integrated fluidic structures and integrated agent reservoirs are shown, tested, and compared in this publication. The single channel setup features a fluidic structure fabricated by anisotropic etching of silicon. To allow for simultaneous release of different agents even though maintaining the same device size, the second type comprises a double channel fluidic structure, fabricated by photolithographic patterning of TMMF. Dispensed droplet volumes are V = 15 pl and V = 10 pl for the silicon and the TMMF based setups, respectively. Utilizing the agent release cartridges, the application in biological assays was demonstrated by hormone-stimulated premature bud formation in Physcomitrella patens and the individual staining of one single L 929 cell within a confluent grown cell culture.

Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells.

PubMed

Semrau, Stefan; Goldmann, Johanna E; Soumillon, Magali; Mikkelsen, Tarjei S; Jaenisch, Rudolf; van Oudenaarden, Alexander

2017-10-23

Gene expression heterogeneity in the pluripotent state of mouse embryonic stem cells (mESCs) has been increasingly well-characterized. In contrast, exit from pluripotency and lineage commitment have not been studied systematically at the single-cell level. Here we measure the gene expression dynamics of retinoic acid driven mESC differentiation from pluripotency to lineage commitment, using an unbiased single-cell transcriptomics approach. We find that the exit from pluripotency marks the start of a lineage transition as well as a transient phase of increased susceptibility to lineage specifying signals. Our study reveals several transcriptional signatures of this phase, including a sharp increase of gene expression variability and sequential expression of two classes of transcriptional regulators. In summary, we provide a comprehensive analysis of the exit from pluripotency and lineage commitment at the single cell level, a potential stepping stone to improved lineage manipulation through timing of differentiation cues.

Discreet charm of the GABAergic bourgeoisie: superconnected cells conduct developmental symphonies.

PubMed

Case, Marianne; Soltesz, Ivan

2009-12-24

In an exciting study in the December 4(th) issue of Science, Bonifazi and colleagues demonstrated the existence and importance of exceedingly rare but unusually richly connected cells in the developing hippocampus. Manipulating the activity of single GABAergic hub cells modulated network activity patterns, demonstrating their importance for coordinating synchronous activity. 2009 Elsevier Inc. All rights reserved.

Development and biological applications of optical tweezers and Raman spectroscopy

NASA Astrophysics Data System (ADS)

Xie, Chang'an

Optical tweezers is a three-dimensional manipulation tool that employs a gradient force that originates from the single highly focused laser beam. Raman spectroscopy is a molecular analytical tool that can give a highly unique "fingerprint" for each substance by measuring the unique vibrations of its molecules. The combination of these two optical techniques offers a new tool for the manipulation and identification of single biological cells and microscopic particles. In this thesis, we designed and implemented a Laser-Tweezers-Raman-Spectroscopy (LTRS) system, also called the Raman-tweezers, for the simultaneous capture and analysis of both biological particles and non-biological particles. We show that microparticles can be conveniently captured at the focus of a laser beam and the Raman spectra of trapped particles can be acquired with high quality. The LTRS system overcomes the intrinsic Brownian motion and cell motility of microparticles in solution and provides a promising tool for in situ identifying suspicious agents. In order to increase the signal to noise ratio, several schemes were employed in LTRS system to reduce the blank noise and the fluorescence signal coming from analytes and the surrounding background. These techniques include near-infrared excitation, optical levitation, confocal microscopy, and frequency-shifted Raman difference. The LTRS system has been applied for the study in cell biology at the single cell level. With the built Raman-tweezers system, we studied the dynamic physiological processes of single living cells, including cell cycle, the transcription and translation of recombinant protein in transgenic yeast cells and the T cell activation. We also studied cell damage and associated biochemical processes in optical traps, UV radiations, and evaluated heating by near-infrared Raman spectroscopy. These studies show that the Raman-tweezers system is feasible to provide rapid and reliable diagnosis of cellular disorders and can be used as a valuable tool to study cellular processes within single living cells or intracellular organelles and may aid research in molecular and cellular biology.

Detection, manipulation and post processing of circulating tumor cells using optical techniques

NASA Astrophysics Data System (ADS)

Bakhtiaridoost, Somayyeh; Habibiyan, Hamidreza; Ghafoorifard, Hassan

2015-12-01

Circulating tumor cells (CTCs) are malignant cells that are derived from a solid tumor in the metastasis stage and are shed into the blood stream. These cells hold great promise to be used as liquid biopsy that is less aggressive than traditional biopsy. Recently, detection and enumeration of these cells has received ever-increasing attention from researchers as a way of early detection of cancer metastasis, determining the effectiveness of treatment and studying the mechanism of formation of secondary tumors. CTCs are found in blood at low concentration, which is a major limitation of isolation and detection of these cells. Over the last few years, multifarious research studies have been conducted on accurate isolation and detection and post processing of CTCs. Among all the proposed systems, microfluidic systems seem to be more attractive for researchers due to their numerous advantages. On the other hand, recent developments in optical methods have made the possibility of cellular studies at single-cell level. Thus, accuracy and efficiency of separation, detection and manipulation of CTCs can be improved using optical techniques. In this review, we describe optical methods that have been used for CTC detection, manipulation and post processing.

Single cell adhesion force measurement for cell viability identification using an AFM cantilever-based micro putter

NASA Astrophysics Data System (ADS)

Shen, Yajing; Nakajima, Masahiro; Kojima, Seiji; Homma, Michio; Kojima, Masaru; Fukuda, Toshio

2011-11-01

Fast and sensitive cell viability identification is a key point for single cell analysis. To address this issue, this paper reports a novel single cell viability identification method based on the measurement of single cell shear adhesion force using an atomic force microscopy (AFM) cantilever-based micro putter. Viable and nonviable yeast cells are prepared and put onto three kinds of substrate surfaces, i.e. tungsten probe, gold and ITO substrate surfaces. A micro putter is fabricated from the AFM cantilever by focused ion beam etching technique. The spring constant of the micro putter is calibrated using the nanomanipulation approach. The shear adhesion force between the single viable or nonviable cell and each substrate is measured using the micro putter based on the nanorobotic manipulation system inside an environmental scanning electron microscope. The adhesion force is calculated based on the deflection of the micro putter beam. The results show that the adhesion force of the viable cell to the substrate is much larger than that of the nonviable cell. This identification method is label free, fast, sensitive and can give quantitative results at the single cell level.

Combining Microinjection and Immunoblotting to Analyze MAP Kinase Phosphorylation in Single Starfish Oocytes and Eggs

NASA Astrophysics Data System (ADS)

Carroll, David J.; Hua, Wei

The starfish oocyte has proven useful for studies involving microinjection because it is relatively large (190 μm) and optically clear. These oocytes are easily obtained from the ovary arrested at prophase of meiosis I, making them useful as a model system for the study of cell cycle-related events. In this chapter, a method for combining microinjection with immunoblotting of single cells is described. Individual starfish oocytes are injected, removed from the microinjection chamber, and analyzed by immunoblotting for the dual-phosphorylated form of mitogen-activated protein kinase (MAPK). This method will allow for experiments testing the regulation of MAPK in single cells and for the manipulation of these cells by a quantitative microinjection technique.

Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping

NASA Astrophysics Data System (ADS)

Labib, Mahmoud; Mohamadi, Reza M.; Poudineh, Mahla; Ahmed, Sharif U.; Ivanov, Ivaylo; Huang, Ching-Lung; Moosavi, Maral; Sargent, Edward H.; Kelley, Shana O.

2018-05-01

Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis—single-cell mRNA cytometry—that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.

An integrated microfluidic cell for detection, manipulation, and sorting of single micron-sized magnetic beads

NASA Astrophysics Data System (ADS)

Jiang, Z.; Llandro, J.; Mitrelias, T.; Bland, J. A. C.

2006-04-01

A lab-on-a-chip integrated microfluidic cell has been developed for magnetic biosensing, which is comprised of anisotropic magnetoresistance (AMR) sensors optimized for the detection of single magnetic beads and electrodes to manipulate and sort the beads, integrated into a microfluidic channel. The device is designed to read out the real-time signal from 9 μm diameter magnetic beads moving over AMR sensors patterned into 18×4.5 μm rectangles and 10 μm diameter rings and arranged in Wheatstone bridges. The beads are moved over the sensors along a 75×75 μm wide channel patterned in SU8. Beads of different magnetic moments can be sorted through a magnetostatic sorting gate into different branches of the microfluidic channel using a magnetic field gradient applied by lithographically defined 120 nm thick Cu striplines carrying 0.2 A current.

Analysis of peripheral B cells and autoantibodies against the anti-nicotinic acetylcholine receptor derived from patients with myasthenia gravis using single-cell manipulation tools

PubMed Central

Terakawa, Maki; Muneoka, Satoshi; Nagahira, Kazuhiro; Nagane, Yuriko; Yamate, Jyoji; Motomura, Masakatsu; Utsugisawa, Kimiaki

2017-01-01

The majority of patients with myasthenia gravis (MG), an organ-specific autoimmune disease, harbor autoantibodies that attack the nicotinic acetylcholine receptor (nAChR-Abs) at the neuromuscular junction of skeletal muscles, resulting in muscle weakness. Single cell manipulation technologies coupled with genetic engineering are very powerful tools to examine T cell and B cell repertoires and the dynamics of adaptive immunity. These tools have been utilized to develop mAbs in parallel with hybridomas, phage display technologies and B-cell immortalization. By applying a single cell technology and novel high-throughput cell-based binding assays, we identified peripheral B cells that produce pathogenic nAChR-Abs in patients with MG. Although anti-nAChR antibodies produced by individual peripheral B cells generally exhibited low binding affinity for the α-subunit of the nAChR and great sequence diversity, a small fraction of these antibodies bound with high affinity to native-structured nAChRs on cell surfaces. B12L, one such Ab isolated here, competed with a rat Ab (mAb35) for binding to the human nAChR and thus considered to recognize the main immunogenic region (MIR). By evaluating the Ab in in vitro cell-based assays and an in vivo rat passive transfer model, B12L was found to act as a pathogenic Ab in rodents and presumably in humans.These findings suggest that B cells in peripheral blood may impact MG pathogenicity. Our methodology can be applied not only to validate pathogenic Abs as molecular target of MG treatment, but also to discover and analyze Ab production systems in other human diseases. PMID:29040265

Analysis of peripheral B cells and autoantibodies against the anti-nicotinic acetylcholine receptor derived from patients with myasthenia gravis using single-cell manipulation tools.

PubMed

Makino, Tomohiro; Nakamura, Ryuichi; Terakawa, Maki; Muneoka, Satoshi; Nagahira, Kazuhiro; Nagane, Yuriko; Yamate, Jyoji; Motomura, Masakatsu; Utsugisawa, Kimiaki

2017-01-01

The majority of patients with myasthenia gravis (MG), an organ-specific autoimmune disease, harbor autoantibodies that attack the nicotinic acetylcholine receptor (nAChR-Abs) at the neuromuscular junction of skeletal muscles, resulting in muscle weakness. Single cell manipulation technologies coupled with genetic engineering are very powerful tools to examine T cell and B cell repertoires and the dynamics of adaptive immunity. These tools have been utilized to develop mAbs in parallel with hybridomas, phage display technologies and B-cell immortalization. By applying a single cell technology and novel high-throughput cell-based binding assays, we identified peripheral B cells that produce pathogenic nAChR-Abs in patients with MG. Although anti-nAChR antibodies produced by individual peripheral B cells generally exhibited low binding affinity for the α-subunit of the nAChR and great sequence diversity, a small fraction of these antibodies bound with high affinity to native-structured nAChRs on cell surfaces. B12L, one such Ab isolated here, competed with a rat Ab (mAb35) for binding to the human nAChR and thus considered to recognize the main immunogenic region (MIR). By evaluating the Ab in in vitro cell-based assays and an in vivo rat passive transfer model, B12L was found to act as a pathogenic Ab in rodents and presumably in humans.These findings suggest that B cells in peripheral blood may impact MG pathogenicity. Our methodology can be applied not only to validate pathogenic Abs as molecular target of MG treatment, but also to discover and analyze Ab production systems in other human diseases.

SINGLE MOLECULE APPROACHES TO BIOLOGY, 2010 GORDON RESEARCH CONFERENCE, JUNE 27-JULY 2, 2010, ITALY

DOE Office of Scientific and Technical Information (OSTI.GOV)

Professor William Moerner

2010-07-09

The 2010 Gordon Conference on Single-Molecule Approaches to Biology focuses on cutting-edge research in single-molecule science. Tremendous technical developments have made it possible to detect, identify, track, and manipulate single biomolecules in an ambient environment or even in a live cell. Single-molecule approaches have changed the way many biological problems are addressed, and new knowledge derived from these approaches continues to emerge. The ability of single-molecule approaches to avoid ensemble averaging and to capture transient intermediates and heterogeneous behavior renders them particularly powerful in elucidating mechanisms of biomolecular machines: what they do, how they work individually, how they work together,more » and finally, how they work inside live cells. The burgeoning use of single-molecule methods to elucidate biological problems is a highly multidisciplinary pursuit, involving both force- and fluorescence-based methods, the most up-to-date advances in microscopy, innovative biological and chemical approaches, and nanotechnology tools. This conference seeks to bring together top experts in molecular and cell biology with innovators in the measurement and manipulation of single molecules, and will provide opportunities for junior scientists and graduate students to present their work in poster format and to exchange ideas with leaders in the field. A number of excellent poster presenters will be selected for short oral talks. Topics as diverse as single-molecule sequencing, DNA/RNA/protein interactions, folding machines, cellular biophysics, synthetic biology and bioengineering, force spectroscopy, new method developments, superresolution imaging in cells, and novel probes for single-molecule imaging will be on the program. Additionally, the collegial atmosphere of this Conference, with programmed discussion sessions as well as opportunities for informal gatherings in the afternoons and evenings in the beauty of the Il Ciocco site in Tuscany, provides an avenue for scientists from different disciplines to interact and brainstorm and promotes cross-disciplinary collaborations directed toward compelling biological problems.« less

Light-neuron interactions: key to understanding the brain

NASA Astrophysics Data System (ADS)

Go, Mary Ann; Daria, Vincent R.

2017-02-01

In recent years, advances in light-based technology have driven an ongoing optical revolution in neuroscience. Synergistic technologies in laser microscopy, molecular biology, organic and synthetic chemistry, genetic engineering and materials science have allowed light to overcome the limitations of and to replace many conventional tools used by physiologists to record from and to manipulate single cells or whole cellular networks. Here we review the different optical techniques for stimulating neurons, influencing neuronal growth, manipulating neuronal structures and neurosurgery.

A Single Multiplex crRNA Array for FnCpf1-Mediated Human Genome Editing.

PubMed

Sun, Huihui; Li, Fanfan; Liu, Jie; Yang, Fayu; Zeng, Zhenhai; Lv, Xiujuan; Tu, Mengjun; Liu, Yeqing; Ge, Xianglian; Liu, Changbao; Zhao, Junzhao; Zhang, Zongduan; Qu, Jia; Song, Zongming; Gu, Feng

2018-06-15

Cpf1 has been harnessed as a tool for genome manipulation in various species because of its simplicity and high efficiency. Our recent study demonstrated that FnCpf1 could be utilized for human genome editing with notable advantages for target sequence selection due to the flexibility of the protospacer adjacent motif (PAM) sequence. Multiplex genome editing provides a powerful tool for targeting members of multigene families, dissecting gene networks, modeling multigenic disorders in vivo, and applying gene therapy. However, there are no reports at present that show FnCpf1-mediated multiplex genome editing via a single customized CRISPR RNA (crRNA) array. In the present study, we utilize a single customized crRNA array to simultaneously target multiple genes in human cells. In addition, we also demonstrate that a single customized crRNA array to target multiple sites in one gene could be achieved. Collectively, FnCpf1, a powerful genome-editing tool for multiple genomic targets, can be harnessed for effective manipulation of the human genome. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

3-dimensional dark traps for low refractive index bio-cells using a single optical fiber Bessel beam.

PubMed

Zhang, Yu; Tang, Xiaoyun; Zhang, Yaxun; Su, Wenjie; Liu, Zhihai; Yang, Xinghua; Zhang, Jianzhong; Yang, Jun; Oh, Kyunghwan; Yuan, Libo

2018-06-15

We proposed and experimentally demonstrated 3-dimensional dark traps for low refractive index bio-cells using a single optical fiber Bessel beam. The Bessel beam was produced by concatenating single-mode fiber and a step index multimode fiber, which was then focused by a high refractive index glass microsphere integrated on the fiber end facet. The focused Bessel beam provided two dark fields along the axial direction, where stable trapping of low refractive index bio-cells was realized in a high refractive index liquid bath. The all-fiber and seamlessly integrated structure of the proposed scheme can find ample potential as a micro-optical probe in in situ characterization and manipulation of multiple bio-cells with refractive indices lower than that of the liquid bath.

Ultra-localized single cell electroporation using silicon nanowires.

PubMed

Jokilaakso, Nima; Salm, Eric; Chen, Aaron; Millet, Larry; Guevara, Carlos Duarte; Dorvel, Brian; Reddy, Bobby; Karlstrom, Amelie Eriksson; Chen, Yu; Ji, Hongmiao; Chen, Yu; Sooryakumar, Ratnasingham; Bashir, Rashid

2013-02-07

Analysis of cell-to-cell variation can further the understanding of intracellular processes and the role of individual cell function within a larger cell population. The ability to precisely lyse single cells can be used to release cellular components to resolve cellular heterogeneity that might be obscured when whole populations are examined. We report a method to position and lyse individual cells on silicon nanowire and nanoribbon biological field effect transistors. In this study, HT-29 cancer cells were positioned on top of transistors by manipulating magnetic beads using external magnetic fields. Ultra-rapid cell lysis was subsequently performed by applying 600-900 mV(pp) at 10 MHz for as little as 2 ms across the transistor channel and the bulk substrate. We show that the fringing electric field at the device surface disrupts the cell membrane, leading to lysis from irreversible electroporation. This methodology allows rapid and simple single cell lysis and analysis with potential applications in medical diagnostics, proteome analysis and developmental biology studies.

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

DOE PAGES

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.; ...

2017-03-13

Mechanical forces in the cell’s natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging frommore » the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques.« less

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

PubMed Central

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.; England, Grant T; Kim, Philseok; Kolle, Mathias; Ferrante, Thomas; Zarzar, Lauren D; Strong, Elizabeth; Aizenberg, Joanna

2017-01-01

Mechanical forces in the cell’s natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging from the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques. PMID:28287116

Photothermally triggered actuation of hybrid materials as a new platform for in vitro cell manipulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sutton, Amy; Shirman, Tanya; Timonen, Jaakko V. I.

Mechanical forces in the cell’s natural environment have a crucial impact on growth, differentiation and behaviour. Few areas of biology can be understood without taking into account how both individual cells and cell networks sense and transduce physical stresses. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here we report a new type of active cell culture material that allows highly localized, directional and reversible deformation of the cell growth substrate, with control at scales ranging frommore » the entire surface to the subcellular, and response times on the order of seconds. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques.« less

Backscattered EM-wave manipulation using low cost 1-bit reflective surface at W-band

NASA Astrophysics Data System (ADS)

Taher Al-Nuaimi, Mustafa K.; Hong, Wei; He, Yejun

2018-04-01

The design of low cost 1-bit reflective (non-absorptive) surfaces for manipulation of backscattered EM-waves and radar cross section (RCS) reduction at W-band is presented in this article. The presented surface is designed based on the reflection phase cancellation principle. The unit cell used to compose the proposed surface has an obelus (division symbol of short wire and two disks above and below) like shape printed on a grounded dielectric material. Using this unit cell, surfaces that can efficiently manipulate the backscattered RCS pattern by using the proposed obelus-shaped unit cell (as ‘0’ element) and its mirrored unit cell (as ‘1’ element) in one surface with a 180°  ±  35° reflection phase difference between their reflection phases are designed. The proposed surfaces can generate various kinds of backscattered RCS patterns, such as single, three, or four lobes or even a low-level (reduced RCS) diffused reflection pattern when those two unit cells are distributed randomly across the surface aperture. For experimental characterization purposes, a 50  ×  50 mm2 surface is fabricated and measured.

Analysis of single mammalian cells on-chip.

PubMed

Sims, Christopher E; Allbritton, Nancy L

2007-04-01

A goal of modern biology is to understand the molecular mechanisms underlying cellular function. The ability to manipulate and analyze single cells is crucial for this task. The advent of microengineering is providing biologists with unprecedented opportunities for cell handling and investigation on a cell-by-cell basis. For this reason, lab-on-a-chip (LOC) technologies are emerging as the next revolution in tools for biological discovery. In the current discussion, we seek to summarize the state of the art for conventional technologies in use by biologists for the analysis of single, mammalian cells, and then compare LOC devices engineered for these same single-cell studies. While a review of the technical progress is included, a major goal is to present the view point of the practicing biologist and the advances that might increase adoption by these individuals. The LOC field is expanding rapidly, and we have focused on areas of broad interest to the biology community where the technology is sufficiently far advanced to contemplate near-term application in biological experimentation. Focus areas to be covered include flow cytometry, electrophoretic analysis of cell contents, fluorescent-indicator-based analyses, cells as small volume reactors, control of the cellular microenvironment, and single-cell PCR.

Magnetophoretic circuits for digital control of single particles and cells

NASA Astrophysics Data System (ADS)

Lim, Byeonghwa; Reddy, Venu; Hu, Xinghao; Kim, Kunwoo; Jadhav, Mital; Abedini-Nassab, Roozbeh; Noh, Young-Woock; Lim, Yong Taik; Yellen, Benjamin B.; Kim, Cheolgi

2014-05-01

The ability to manipulate small fluid droplets, colloidal particles and single cells with the precision and parallelization of modern-day computer hardware has profound applications for biochemical detection, gene sequencing, chemical synthesis and highly parallel analysis of single cells. Drawing inspiration from general circuit theory and magnetic bubble technology, here we demonstrate a class of integrated circuits for executing sequential and parallel, timed operations on an ensemble of single particles and cells. The integrated circuits are constructed from lithographically defined, overlaid patterns of magnetic film and current lines. The magnetic patterns passively control particles similar to electrical conductors, diodes and capacitors. The current lines actively switch particles between different tracks similar to gated electrical transistors. When combined into arrays and driven by a rotating magnetic field clock, these integrated circuits have general multiplexing properties and enable the precise control of magnetizable objects.

CalQuo: automated, simultaneous single-cell and population-level quantification of global intracellular Ca2+ responses.

PubMed

Fritzsche, Marco; Fernandes, Ricardo A; Colin-York, Huw; Santos, Ana M; Lee, Steven F; Lagerholm, B Christoffer; Davis, Simon J; Eggeling, Christian

2015-11-13

Detecting intracellular calcium signaling with fluorescent calcium indicator dyes is often coupled with microscopy techniques to follow the activation state of non-excitable cells, including lymphocytes. However, the analysis of global intracellular calcium responses both at the single-cell level and in large ensembles simultaneously has yet to be automated. Here, we present a new software package, CalQuo (Calcium Quantification), which allows the automated analysis and simultaneous monitoring of global fluorescent calcium reporter-based signaling responses in up to 1000 single cells per experiment, at temporal resolutions of sub-seconds to seconds. CalQuo quantifies the number and fraction of responding cells, the temporal dependence of calcium signaling and provides global and individual calcium-reporter fluorescence intensity profiles. We demonstrate the utility of the new method by comparing the calcium-based signaling responses of genetically manipulated human lymphocytic cell lines.

Plant Development & the Fern Life Cycle: Using "Ceratopteris richardii."

ERIC Educational Resources Information Center

Renzaglia, Karen S.; And Others

1995-01-01

Presents laboratory activities focusing on the development of sexually mature gametophytes from single-celled spores. Includes techniques for culture and manipulation of gametophyte development from spores that are applicable for hands-on activities for students at all levels. (MKR)

Identification and optogenetic manipulation of memory engrams in the hippocampus

PubMed Central

Ramirez, Steve; Tonegawa, Susumu; Liu, Xu

2014-01-01

With the accumulation of our knowledge about how memories are formed, consolidated, retrieved, and updated, neuroscience is now reaching a point where discrete memories can be identified and manipulated at rapid timescales. Here, we start with historical studies that lead to the modern memory engram theory. Then, we will review recent advances in memory engram research that combine transgenic and optogenetic approaches to reveal the underlying neuronal substrates sufficient for activating mnemonic processes. We will focus on three concepts: (1) isolating memory engrams at the level of single cells to tag them for subsequent manipulation; (2) testing the sufficiency of these engrams for memory recall by artificially activating them; and (3) presenting new stimuli during the artificial activation of these engrams to induce an association between the two to form a false memory. We propose that hippocampal cells that show activity-dependent changes during learning construct a cellular basis for contextual memory engrams. PMID:24478647

Engineering the Intracellular Micro- and Nano-environment via Magnetic Nanoparticles

NASA Astrophysics Data System (ADS)

Tseng, Peter

Single cells, despite being the base unit of living organisms, possess a high degree of hierarchical structure and functional compartmentalization. This complexity exists for good reason: cells must respond efficiently and effectively to its surrounding environment by differentiating, moving, interacting, and more in order to survive or inhabit its role in the larger biological system. At the core of these responses is cellular decision-making. Cells process cues internally and externally from the environment and effect intracellular asymmetry in biochemistry and structure in order to carry out the proper biological responses. Functionalized magnetic particles have shown to be a powerful tool in interacting with biological matter, through either cell or biomolecule sorting, and the activation of biological processes. This dissertation reports on techniques utilizing manipulated magnetic nanoparticles (internalized by cells) to spatially and temporally localize intracellular cues, and examines the resulting asymmetry in biological processes generated by our methods. We first examine patterned micromagnetic elements as a simple strategy of rapidly manipulating magnetic nanoparticles throughout the intracellular space. Silicon or silicon dioxide substrates form the base for electroplated NiFe rods, which are repeated at varying size and pitch. A planarizing resin, initially SU-8, is used as the substrate layer for cellular adhesion. We demonstrate that through the manipulations of a simple external magnet, these micro-fabricated substrates can mediate rapid (under 2 s) and precise (submicron), reversible translation of magnetic nanoparticles through cellular space. Seeding cells on substrates composed of these elements allows simultaneous control of ensembles of nanoparticles over thousands of cells at a time. We believe such substrates could form the basis of magnetically based tools for the activation of biological matter. We further utilize these strategies to generate user-controllable (time-varying and localizable), massively parallel forces on arrays of cells mediated by coalesced ensembles of magnetic nanoparticles. The above process is simplified and adapted for single cell analysis by precisely aligning fibronectin patterned cells to a single flanking micromagnet. The cells are loaded with magnetic-fluorescent nanoparticles, which are then localized to uniform positions at the internal edge of the cell membrane over huge arrays of cells using large external fields, allowing us to conduct composed studies on cellular response to force. By applying forces approaching the yield tension (5 nN / mum) of single cells, we are able to generate highly coordinated responses in cellular behavior. We discover that increasing tension generates highly directed, PAK-dependent leading-edge type filopodia that increase in intensity with rising tension. In addition, we find that our generated forces can simulate cues created during cellular mitosis, as we are consistently able to generate significant (45 to 90 degree) biasing of the metaphase plate during cell division. Large sample size and rapid sample generation also allow us to analyze cells at an unprecedented rate---a single sample can simultaneously stimulate thousands of cells for high statistical accuracy in measurements. We believe these approaches have potential not just as a tool to study single-cell response, but as a means of cell control, potentially through modifying cell movement, division, or differentiation. More generally, once approaches to release nanoparticles from endosomes are implemented, the technique provides a platform to dynamically apply a range of localized stimuli arbitrarily within cells. Through the bioconjugation of proteins, nucleic acids, small molecules, or whole organelles a broad range of questions should be accessible concerning molecular localization and its importance in cell function.

Detection of Her2-overexpressing cancer cells using keyhole shaped chamber array employing a magnetic droplet-handling system.

PubMed

Okochi, Mina; Koike, Shinji; Tanaka, Masayoshi; Honda, Hiroyuki

2017-07-15

An on-chip gene expression analysis compartmentalized in droplets was developed for detection of cancer cells at a single-cell level. The chip consists of a keyhole-shaped reaction chamber with hydrophobic modification employing a magnetic bead-droplet-handling system with a gate for bead separation. Using three kinds of water-based droplets in oil, a droplet with sample cells, a lysis buffer with magnetic beads, and RT-PCR buffer, parallel magnetic manipulation and fusion of droplets were performed using a magnet-handling device containing small external magnet patterns in an array. The actuation with the magnet offers a simple system for droplet manipulation that allows separation and fusion of droplets containing magnetic beads. After reverse transcription and amplification by thermal cycling, fluorescence was obtained for detection of overexpressing genes. For clinical detection of gastric cancer cells in peritoneal washing, the Her2-overexpressing gastric cancer cells spiked within normal cells was detected by gene expression analysis of droplets containing an average of 2.5 cells. Our developed droplet-based cancer detection system manipulated by external magnetic force without pumps or valves offers a simple and flexible set-up for transcriptional detection of cancer cells, and will be greatly advantageous for less-invasive clinical diagnosis and prognostic prediction. Copyright © 2016 Elsevier B.V. All rights reserved.

A robust and tunable mitotic oscillator in artificial cells

PubMed Central

Wang, Shiyuan; Barnes, Patrick M; Liu, Xuwen; Xu, Haotian; Jin, Minjun; Liu, Allen P

2018-01-01

Single-cell analysis is pivotal to deciphering complex phenomena like heterogeneity, bistability, and asynchronous oscillations, where a population ensemble cannot represent individual behaviors. Bulk cell-free systems, despite having unique advantages of manipulation and characterization of biochemical networks, lack the essential single-cell information to understand a class of out-of-steady-state dynamics including cell cycles. Here, by encapsulating Xenopus egg extracts in water-in-oil microemulsions, we developed artificial cells that are adjustable in sizes and periods, sustain mitotic oscillations for over 30 cycles, and function in forms from the simplest cytoplasmic-only to the more complicated ones involving nuclear dynamics, mimicking real cells. Such innate flexibility and robustness make it key to studying clock properties like tunability and stochasticity. Our results also highlight energy as an important regulator of cell cycles. We demonstrate a simple, powerful, and likely generalizable strategy of integrating strengths of single-cell approaches into conventional in vitro systems to study complex clock functions. PMID:29620527

Functional mapping of cell surface proteins with localized stimulation of single cells

NASA Astrophysics Data System (ADS)

Sun, Bingyun; Chiu, Daniel T.

2003-11-01

This paper describes the development of using individual micro and nano meter-sized vesicles as delivery vessels to functionally map the distribution of cell surface proteins at the level of single cells. The formation of different sizes of vesicles from tens of nanometers to a few micrometers in diameter that contain the desired molecules is addressed. An optical trap is used to manipulate the loaded vesicle to specific cell morphology of interest, and a pulsed UV laser is used to photo-release the stimuli onto the cell membrane. Carbachol activated cellular calcium flux, upon binding to muscarinic acetylcholine receptors, is studied by this method, and the potential of using this method for the functional mapping of localized proteins on the cell surface membrane is discussed.

Exclusive Liquid Repellency: An Open Multi-Liquid-Phase Technology for Rare Cell Culture and Single-Cell Processing.

PubMed

Li, Chao; Yu, Jiaquan; Schehr, Jennifer; Berry, Scott M; Leal, Ticiana A; Lang, Joshua M; Beebe, David J

2018-05-23

The concept of high liquid repellency in multi-liquid-phase systems (e.g., aqueous droplets in an oil background) has been applied to areas of biomedical research to realize intrinsic advantages not available in single-liquid-phase systems. Such advantages have included minimizing analyte loss, facile manipulation of single-cell samples, elimination of biofouling, and ease of use regarding loading and retrieving of the sample. In this paper, we present generalized design rules for predicting the wettability of solid-liquid-liquid systems (especially for discrimination between exclusive liquid repellency (ELR) and finite liquid repellency) to extend the applications of ELR. We then apply ELR to two model systems with open microfluidic design in cell biology: (1) in situ underoil culture and combinatorial coculture of mammalian cells in order to demonstrate directed single-cell multiencapsulation with minimal waste of samples as compared to stochastic cell seeding and (2) isolation of a pure population of circulating tumor cells, which is required for certain downstream analyses including sequencing and gene expression profiling.

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

PubMed Central

Baars, Destiny L.; Pelegri, Francisco

2016-01-01

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species. PMID:28060351

Droplet microfluidic technology for single-cell high-throughput screening.

PubMed

Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J Brian; Rothberg, Jonathan M; Link, Darren R; Perrimon, Norbert; Samuels, Michael L

2009-08-25

We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses.

A novel method for multiparameter physiological phenotype characterization at the single-cell level

NASA Astrophysics Data System (ADS)

Kelbauskas, Laimonas; Ashili, Shashanka; Houkal, Jeff; Smith, Dean; Mohammadreza, Aida; Lee, Kristen; Kumar, Ashok; Anis, Yasser; Paulson, Tom; Youngbull, Cody; Tian, Yanqing; Johnson, Roger; Holl, Mark; Meldrum, Deirdre

2011-02-01

Non-genetic intercellular heterogeneity has been increasingly recognized as one of the key factors in a variety of core cellular processes including proliferation, stimulus response, carcinogenesis and drug resistance. Many diseases, including cancer, originate in a single or a few cells. Early detection and characterization of these abnormal cells can provide new insights into the pathogenesis and serve as a tool for better disease diagnosis and treatment. We report on a novel technology for multiparameter physiological phenotype characterization at the single-cell level. It is based on real-time measurements of concentrations of several metabolites by means of extracellular optical sensors in microchambers of sub-nL volume containing single cells. In its current configuration, the measurement platform features the capability to detect oxygen consumption rate and pH changes under normoxic and hypoxic conditions at the single-cell level. We have conceived, designed and developed a semi-automated method for single-cell manipulation and loading into microwells utilizing custom, high-precision fluid handling at the nanoliter scale. We present the results of a series of measurements of oxygen consumption rates (OCRs) of single human metaplastic esophageal epithelial cells. In addition, to assess the effects of cell-to-cell interactions, we have measured OCRs of two and three cells placed in a single well. The major advantages of the approach are a) multiplexed characterization of cell phenotype at the single-cell level, b) minimal invasiveness due to the distant positioning of sensors, and c) flexibility in terms of accommodating measurements of other metabolites or biomolecules of interest.

Neural stem cell heterogeneity through time and space in the ventricular-subventricular zone.

PubMed

Rushing, Gabrielle; Ihrie, Rebecca A

2016-08-01

The origin and classification of neural stem cells (NSCs) has been a subject of intense investigation for the past two decades. Efforts to categorize NSCs based on their location, function and expression have established that these cells are a heterogeneous pool in both the embryonic and adult brain. The discovery and additional characterization of adult NSCs has introduced the possibility of using these cells as a source for neuronal and glial replacement following injury or disease. To understand how one could manipulate NSC developmental programs for therapeutic use, additional work is needed to elucidate how NSCs are programmed and how signals during development are interpreted to determine cell fate. This review describes the identification, classification and characterization of NSCs within the large neurogenic niche of the ventricular-subventricular zone (V-SVZ). A literature search was conducted using Pubmed including the keywords "ventricular-subventricular zone," "neural stem cell," "heterogeneity," "identity" and/or "single cell" to find relevant manuscripts to include within the review. A special focus was placed on more recent findings using single-cell level analyses on neural stem cells within their niche(s). This review discusses over 20 research articles detailing findings on V-SVZ NSC heterogeneity, over 25 articles describing fate determinants of NSCs, and focuses on 8 recent publications using distinct single-cell analyses of neural stem cells including flow cytometry and RNA-seq. Additionally, over 60 manuscripts highlighting the markers expressed on cells within the NSC lineage are included in a chart divided by cell type. Investigation of NSC heterogeneity and fate decisions is ongoing. Thus far, much research has been conducted in mice however, findings in human and other mammalian species are also discussed here. Implications of NSC heterogeneity established in the embryo for the properties of NSCs in the adult brain are explored, including how these cells may be redirected after injury or genetic manipulation.

Microfluidics cell sample preparation for analysis: Advances in efficient cell enrichment and precise single cell capture

PubMed Central

Bian, Shengtai; Cheng, Yinuo; Shi, Guanya; Liu, Peng; Ye, Xiongying

2017-01-01

Single cell analysis has received increasing attention recently in both academia and clinics, and there is an urgent need for effective upstream cell sample preparation. Two extremely challenging tasks in cell sample preparation—high-efficiency cell enrichment and precise single cell capture—have now entered into an era full of exciting technological advances, which are mostly enabled by microfluidics. In this review, we summarize the category of technologies that provide new solutions and creative insights into the two tasks of cell manipulation, with a focus on the latest development in the recent five years by highlighting the representative works. By doing so, we aim both to outline the framework and to showcase example applications of each task. In most cases for cell enrichment, we take circulating tumor cells (CTCs) as the target cells because of their research and clinical importance in cancer. For single cell capture, we review related technologies for many kinds of target cells because the technologies are supposed to be more universal to all cells rather than CTCs. Most of the mentioned technologies can be used for both cell enrichment and precise single cell capture. Each technology has its own advantages and specific challenges, which provide opportunities for researchers in their own area. Overall, these technologies have shown great promise and now evolve into real clinical applications. PMID:28217240

Photothermal gold nanoparticle mediated stimulation of cardiomyocyte beating (Conference Presentation)

NASA Astrophysics Data System (ADS)

Kalies, Stefan; Gentemann, Lara; Coffee, Michelle; Zweigerdt, Robert; Heinemann, Dag; Heisterkamp, Alexander

2017-03-01

Photothermal manipulation of cells via heating of gold nanoparticles has proven to be an efficient tool for molecular delivery into cells via cell perforation with short laser pulses. We investigated a potential extension of this technique for cell stimulation of cardiomyocytes using a 532 nm and 850 ps laser system and a surface concentration of 0.5 μg/cm2 of 200 nm gold nanoparticles. The gold nanoparticles were unspecifically attached to the cardiomyocytes after an incubation period of three hours. The laser irradiation leads to a temperature rise directly at the particles of several hundred degrees K which evokes bubble formation and membrane perforation. We examined the effect of laser based photothermal manipulation at different laser powers, with different calcium concentrations, and for a cardiomyocyte-like cell line (HL1 cells), neonatal rat cardiomyocytes and human embryonic stem cell (hESC)-derived cardiomyocytes. Fast calcium oscillations in HL1 cells were observed in the presence and absence of extracellular calcium and most pronounced in the area next to the laser spot after irradiation. Within the laser spot, in particular high laser powers led to a single rise in calcium over a time period of several seconds. These results were confirmed in stem cell-derived cardiomyocytes. In the presence of normal and high calcium concentrations, the spontaneous contraction frequency increased after laser irradiation in neonatal rat cardiomyocytes. Consequently, gold nanoparticle mediated photothermal cell manipulation via pulsed lasers may serve as a potential pacemaker-technique in regenerative approaches, next to optogenetics.

Manipulating Ion Migration for Highly Stable Light-Emitting Diodes with Single-Crystalline Organometal Halide Perovskite Microplatelets.

PubMed

Chen, Mingming; Shan, Xin; Geske, Thomas; Li, Junqiang; Yu, Zhibin

2017-06-27

Ion migration has been commonly observed as a detrimental phenomenon in organometal halide perovskite semiconductors, causing the measurement hysteresis in solar cells and ultrashort operation lifetimes in light-emitting diodes. In this work, ion migration is utilized for the formation of a p-i-n junction at ambient temperature in single-crystalline organometal halide perovskites. The junction is subsequently stabilized by quenching the ionic movement at a low temperature. Such a strategy of manipulating the ion migration has led to efficient single-crystalline light-emitting diodes that emit 2.3 eV photons starting at 1.8 V and sustain a continuous operation for 54 h at ∼5000 cd m -2 without degradation of brightness. In addition, a whispering-gallery-mode cavity and exciton-exciton interaction in the perovskite microplatelets have both been observed that can be potentially useful for achieving electrically driven laser diodes based on single-crystalline organometal halide perovskite semiconductors.

Nanopipette Apparatus for Manipulating Cells

NASA Technical Reports Server (NTRS)

Vilozny, Boaz (Inventor); Seger, R. Adam (Inventor); Actis, Paolo (Inventor); Pourmand, Nader (Inventor)

2017-01-01

Disclosed herein are methods and systems for controlled ejection of desired material onto surfaces including in single cells using nanopipettes, as well as ejection onto and into cells. Some embodiments are directed to a method and system comprising nanopipettes combined with an xyz controller for depositing a user defined pattern on an arbitrary substrate for the purpose of controlled cell adhesion and growth. Alternate embodiments are directed to a method and system comprising nanopipettes combined with an xyz controller and electronic control of a voltage differential in a bore of the nanopipette electroosmotically injecting material into a cell in a high-throughput manner and with minimal damage to the cell. Yet other embodiments are directed to method and system comprising functionalized nanopipettes combined with scanning ion conductance microscopy for studying molecular interactions and detection of biomolecules inside a single living cell.

Lab-on-a-chip technologies for proteomic analysis from isolated cells.

PubMed

Sedgwick, H; Caron, F; Monaghan, P B; Kolch, W; Cooper, J M

2008-10-06

Lab-on-a-chip systems offer a versatile environment in which low numbers of cells and molecules can be manipulated, captured, detected and analysed. We describe here a microfluidic device that allows the isolation, electroporation and lysis of single cells. A431 human epithelial carcinoma cells, expressing a green fluorescent protein-labelled actin, were trapped by dielectrophoresis within an integrated lab-on-a-chip device containing saw-tooth microelectrodes. Using these same trapping electrodes, on-chip electroporation was performed, resulting in cell lysis. Protein release was monitored by confocal fluorescence microscopy.

3D Nanochannel Array Platform for High-throughput Cell Manipulation and Nano-electroporation

NASA Astrophysics Data System (ADS)

Chang, Lingqian

Electroporation is one of the most common non-viral methods for gene delivery. Recent progress in gene therapy has offered special opportunities to electroporation for in vitro and in vivo applications. However, conventional bulk electroporation (BEP) inevitably causes serious cell damage and stochastic transfection between cells. Microfluidic electroporation (MEP) has been claimed to provide benign single cell transfection for the last decade. Nevertheless, the intracellular transport in both MEP and BEP systems is highly diffusion-dominant, which prevents precise dose control and high uniformity. In this Ph.D. research, we developed a 3D nanochannel-electroporation (3D NEP) platform for mass cell transfection. A silicon-based nanochannel array (3D NEP) chip was designed and fabricated for cell manipulation and electroporation. The chip, designed as Z-directional microchannel - nanochannel array, was fabricated by clean room techniques including projection photolithography and deep reactive-ion etching (DRIE). The fabricated 3D NEP chip is capable of handling 40,000 cells per 1 cm2, up to 1 million per wafer (100 mm diameter). High-throughput cell manipulation technologies were investigated for precise alignment of individual cells to the nanochannel array, a key step for NEP to achieve dose control. We developed three techniques for cell trapping in this work. (1) Magnetic tweezers (MTs) were integrated on the chip to remotely control cells under a programmed magnetic field. (2) A positive dielectrophoresis (pDEP) power system was built as an alternative to trap cells onto the nanochannel array using DEP force. (3) A novel yet simple 'dipping-trap' method was used to rapidly trap cells onto a nanochannel array, aligned by a micro-cap array pattern on the 3D NEP chip, which eventually offered 70 - 90 % trapping efficiency and 90 % specificity. 3D NEP platforms were assembled for cell transfection based on the Si-based nanochannel array chip and cell manipulation techniques. Cells were patterned on the nanochannel array and collectively were electroporated in parallel, injected with cargo in Z-direction. Controlling the dose was demonstrated with the external pulse durations at high-throughput. The 'electrophoretic'- expedited delivery of large molecular weight plasmids were demonstrated with large numbers of primary cells simultaneously, which cannot be achieved in BEP and MEP. Two clinically valuable case studies were performed with our 3D NEP for living cell sensing / interrogation. (1) In the case of in vitro transfection of primary cardiomyocytes, we studied the dose-effects of miR-29 on mitochondrial changes and the suppression of the Mcl-1 gene in adult mouse cardiomyocytes by precisely controlling the miR-29 dose injected. (2) Glioma stem cells (GSCs), a type of cell hypothesized to be highly aggressive and to lead to the relapses of gliobastoma in human brain, was studied at single cell resolution on 3D NEP platform. The developed 3D NEP system moves towards clinically oriented and user-friendly tools for life science applications. The batch-treated cells with controlled dosage delivery provide a useful tool for single cell analysis. The pioneering experiments in this work have demonstrated the 3D NEP for the applications of cell reprogramming, adoptive immunotherapy, in vitro cardiomyocytes transfection and glioma stem cells study.

Manipulation of cell membrane using carbon nanotube scaffold as a photoresponsive stimuli generator.

PubMed

Sada, Takao; Fujigaya, Tsuyohiko; Nakashima, Naotoshi

2014-08-01

We describe, for the first time, the perforation of the cell membrane in the targeted single cell based on the nanosecond pulsed near-infrared (NIR) laser irradiation of a thin film of carbon nanotubes that act as an effective photon absorber as well as stimuli generator. When the power of NIR laser is over 17.5 μ J/pulse, the cell membrane after irradiation is irreversibly disrupted and results in cell death. In sharp contrast, the perforation of the cell membrane occurs at suitable laser power (∼15 μ J/pulse) without involving cell termination.

Laser-induced fusion of human embryonic stem cells with optical tweezers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Shuxun; Wang Xiaolin; Sun Dong

2013-07-15

We report a study on the laser-induced fusion of human embryonic stem cells (hESCs) at the single-cell level. Cells were manipulated by optical tweezers and fused under irradiation with pulsed UV laser at 355 nm. Successful fusion was indicated by green fluorescence protein transfer. The influence of laser pulse energy on the fusion efficiency was investigated. The fused products were viable as gauged by live cell staining. Successful fusion of hESCs with somatic cells was also demonstrated. The reported fusion outcome may facilitate studies of cell differentiation, maturation, and reprogramming.

Manipulation of cell membrane using carbon nanotube scaffold as a photoresponsive stimuli generator

PubMed Central

Sada, Takao; Fujigaya, Tsuyohiko; Nakashima, Naotoshi

2014-01-01

We describe, for the first time, the perforation of the cell membrane in the targeted single cell based on the nanosecond pulsed near-infrared (NIR) laser irradiation of a thin film of carbon nanotubes that act as an effective photon absorber as well as stimuli generator. When the power of NIR laser is over 17.5 μJ/pulse, the cell membrane after irradiation is irreversibly disrupted and results in cell death. In sharp contrast, the perforation of the cell membrane occurs at suitable laser power (∼15 μJ/pulse) without involving cell termination. PMID:27877703

Combining single-molecule manipulation and single-molecule detection.

PubMed

Cordova, Juan Carlos; Das, Dibyendu Kumar; Manning, Harris W; Lang, Matthew J

2014-10-01

Single molecule force manipulation combined with fluorescence techniques offers much promise in revealing mechanistic details of biomolecular machinery. Here, we review force-fluorescence microscopy, which combines the best features of manipulation and detection techniques. Three of the mainstay manipulation methods (optical traps, magnetic traps and atomic force microscopy) are discussed with respect to milestones in combination developments, in addition to highlight recent contributions to the field. An overview of additional strategies is discussed, including fluorescence based force sensors for force measurement in vivo. Armed with recent exciting demonstrations of this technology, the field of combined single-molecule manipulation and single-molecule detection is poised to provide unprecedented views of molecular machinery. Copyright © 2014 Elsevier Ltd. All rights reserved.

Droplet-based gene expression analysis using a device with magnetic force-based-droplet-handling system.

PubMed

Okochi, Mina; Tsuchiya, Hiroyoshi; Kumazawa, Fumitaka; Shikida, Mitsuhiro; Honda, Hiroyuki

2010-02-01

A droplet-based cell lysis and reverse transcription-polymerase chain reaction (PCR) were performed on-chip employing magnetic force-based-droplet-handling system. The actuation with a magnet offers a simple system for droplet manipulation; it does not need mechanical fluidic systems such as pumps and valves for handling solutions. It can be used as a powerful tool for various biochemical applications by moving and coalescing sample droplets using magnetic beads immersed in mineral oil. The droplet containing magnetic beads and the cells were manipulated with the magnet located underneath the channel, and coalesced with a droplet of lysis buffer. Using K562 cells as the leukemia model, the cell lysis, cDNA synthesis, and amplification of WT1 gene that is known as the prognostic factor for acute leukemia were successfully performed from a single cell. Copyright (c) 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Microfluidic cell isolation technology for drug testing of single tumor cells and their clusters.

PubMed

Bithi, Swastika S; Vanapalli, Siva A

2017-02-02

Drug assays with patient-derived cells such as circulating tumor cells requires manipulating small sample volumes without loss of rare disease-causing cells. Here, we report an effective technology for isolating and analyzing individual tumor cells and their clusters from minute sample volumes using an optimized microfluidic device integrated with pipettes. The method involves using hand pipetting to create an array of cell-laden nanoliter-sized droplets immobilized in a microfluidic device without loss of tumor cells during the pipetting process. Using this technology, we demonstrate single-cell analysis of tumor cell response to the chemotherapy drug doxorubicin. We find that even though individual tumor cells display diverse uptake profiles of the drug, the onset of apoptosis is determined by accumulation of a critical intracellular concentration of doxorubicin. Experiments with clusters of tumor cells compartmentalized in microfluidic drops reveal that cells within a cluster have higher viability than their single-cell counterparts when exposed to doxorubicin. This result suggests that circulating tumor cell clusters might be able to better survive chemotherapy drug treatment. Our technology is a promising tool for understanding tumor cell-drug interactions in patient-derived samples including rare cells.

The Age of Enlightenment: Evolving Opportunities in Brain Research Through Optical Manipulation of Neuronal Activity

PubMed Central

Jerome, Jason; Heck, Detlef H.

2011-01-01

Optical manipulation of neuronal activity has rapidly developed into the most powerful and widely used approach to study mechanisms related to neuronal connectivity over a range of scales. Since the early use of single site uncaging to map network connectivity, rapid technological development of light modulation techniques has added important new options, such as fast scanning photostimulation, massively parallel control of light stimuli, holographic uncaging, and two-photon stimulation techniques. Exciting new developments in optogenetics complement neurotransmitter uncaging techniques by providing cell-type specificity and in vivo usability, providing optical access to the neural substrates of behavior. Here we review the rapid evolution of methods for the optical manipulation of neuronal activity, emphasizing crucial recent developments. PMID:22275886

The age of enlightenment: evolving opportunities in brain research through optical manipulation of neuronal activity.

PubMed

Jerome, Jason; Heck, Detlef H

2011-01-01

Optical manipulation of neuronal activity has rapidly developed into the most powerful and widely used approach to study mechanisms related to neuronal connectivity over a range of scales. Since the early use of single site uncaging to map network connectivity, rapid technological development of light modulation techniques has added important new options, such as fast scanning photostimulation, massively parallel control of light stimuli, holographic uncaging, and two-photon stimulation techniques. Exciting new developments in optogenetics complement neurotransmitter uncaging techniques by providing cell-type specificity and in vivo usability, providing optical access to the neural substrates of behavior. Here we review the rapid evolution of methods for the optical manipulation of neuronal activity, emphasizing crucial recent developments.

Hydrogen Storage | Hydrogen and Fuel Cells | NREL

Science.gov Websites

research. An International Multi-Laboratory Investigation of Carbon-Based Hydrogen Sorbent Materials Carbon Nanotube Anions, Journal of Materials Research (2012) Manipulation of Hydrogen Binding Energy and Spectroscopy, Journal of Physical Chemistry C (2012) Reactions and Reversible Hydrogenation of Single-Walled

Genetically Encoded Photoactuators and Photosensors for Characterization and Manipulation of Pluripotent Stem Cells

PubMed Central

Pomeroy, Jordan E.; Nguyen, Hung X.; Hoffman, Brenton D.; Bursac, Nenad

2017-01-01

Our knowledge of pluripotent stem cell biology has advanced considerably in the past four decades, but it has yet to deliver on the great promise of regenerative medicine. The slow progress can be mainly attributed to our incomplete understanding of the complex biologic processes regulating the dynamic developmental pathways from pluripotency to fully-differentiated states of functional somatic cells. Much of the difficulty arises from our lack of specific tools to query, or manipulate, the molecular scale circuitry on both single-cell and organismal levels. Fortunately, the last two decades of progress in the field of optogenetics have produced a variety of genetically encoded, light-mediated tools that enable visualization and control of the spatiotemporal regulation of cellular function. The merging of optogenetics and pluripotent stem cell biology could thus be an important step toward realization of the clinical potential of pluripotent stem cells. In this review, we have surveyed available genetically encoded photoactuators and photosensors, a rapidly expanding toolbox, with particular attention to those with utility for studying pluripotent stem cells. PMID:28912894

Systems Analysis of Guard Cell Membrane Transport for Enhanced Stomatal Dynamics and Water Use Efficiency1[W][OPEN

PubMed Central

Wang, Yizhou; Hills, Adrian; Blatt, Michael R.

2014-01-01

Stomatal transpiration is at the center of a crisis in water availability and crop production that is expected to unfold over the next 20 to 30 years. Global water usage has increased 6-fold in the past 100 years, twice as fast as the human population, and is expected to double again before 2030, driven mainly by irrigation and agriculture. Guard cell membrane transport is integral to controlling stomatal aperture and offers important targets for genetic manipulation to improve crop performance. However, its complexity presents a formidable barrier to exploring such possibilities. With few exceptions, mutations that increase water use efficiency commonly have been found to do so with substantial costs to the rate of carbon assimilation, reflecting the trade-off in CO2 availability with suppressed stomatal transpiration. One approach yet to be explored in detail relies on quantitative systems analysis of the guard cell. Our deep knowledge of transport and homeostasis in these cells gives real substance to the prospect for reverse engineering of stomatal responses, using in silico design in directing genetic manipulation for improved water use and crop yields. Here we address this problem with a focus on stomatal kinetics, taking advantage of the OnGuard software and models of the stomatal guard cell recently developed for exploring stomatal physiology. Our analysis suggests that manipulations of single transporter populations are likely to have unforeseen consequences. Channel gating, especially of the dominant K+ channels, appears the most favorable target for experimental manipulation. PMID:24596330

Single-cell intracellular nano-pH probes.

PubMed

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.

Telescience testbed experiments for biomedical studies: fertilization potential recording of amphibian eggs using tele-manipulation under stereoscopic vision.

PubMed

Watanabe, S; Tanaka, M; Wada, Y; Suzuki, H; Takagi, S; Mori, S; Fukai, K; Kanazawa, Y; Takagi, M; Hirakawa, K; Ogasawara, K; Tsumura, K; Ogawa, K; Matsumoto, K; Nagaoka, S; Suzuki, T; Shimura, D; Yamashita, M; Nishio, S

1994-07-01

The telescience testbed experiments were carried out to test and investigate the tele-manipulation techniques in the intracellular potential recording of amphibian eggs. Implementation of telescience testbed was set up in the two separated laboratories of the Tsukuba Space center of NASDA, which were connected by tele-communication links. Manipulators respective for a microelectrode and a sample stage of microscope were moved by computers, of which command signals were transmitted from a computer in a remote control room. The computer in the control room was operated by an investigator (PI) who controlled the movement of each manipulator remotely. A stereoscopic vision of the microscope image were prepared by using a head mounted display (HMD) and were indispensable to the intracellular single cell recording. The fertilization potential of amphibian eggs was successfully obtained through the remote operating system.

Optical levitation and manipulation of stuck particles with pulsed optical tweezers.

PubMed

Ambardekar, Amol Ashok; Li, Yong-Qing

2005-07-15

We report on optical levitation and manipulation of microscopic particles that are stuck on a glass surface with pulsed optical tweezers. An infrared pulse laser at 1.06 microm was used to generate a large gradient force (up to 10(-9) N) within a short duration (approximately 45 micros) that overcomes the adhesive interaction between the particles and the glass surface. Then a low-power continuous-wave diode laser at 785 nm was used to capture and manipulate the levitated particle. We have demonstrated that both stuck dielectric and biological micrometer-sized particles, including polystyrene beads, yeast cells, and Bacillus cereus bacteria, can be levitated and manipulated with this technique. We measured the single-pulse levitation efficiency for 2.0 microm polystyrene beads as a function of the pulse energy and of the axial displacement from the stuck particle to the pulsed laser focus, which was as high as 88%.

A droplet-to-digital (D2D) microfluidic device for single cell assays.

PubMed

Shih, Steve C C; Gach, Philip C; Sustarich, Jess; Simmons, Blake A; Adams, Paul D; Singh, Seema; Singh, Anup K

2015-01-07

We have developed a new hybrid droplet-to-digital microfluidic platform (D2D) that integrates droplet-in-channel microfluidics with digital microfluidics (DMF) for performing multi-step assays. This D2D platform combines the strengths of the two formats-droplets-in-channel for facile generation of droplets containing single cells, and DMF for on-demand manipulation of droplets including control of different droplet volumes (pL-μL), creation of a dilution series of ionic liquid (IL), and parallel single cell culturing and analysis for IL toxicity screening. This D2D device also allows for automated analysis that includes a feedback-controlled system for merging and splitting of droplets to add reagents, an integrated Peltier element for parallel cell culture at optimum temperature, and an impedance sensing mechanism to control the flow rate for droplet generation and preventing droplet evaporation. Droplet-in-channel is well-suited for encapsulation of single cells as it allows the careful manipulation of flow rates of aqueous phase containing cells and oil to optimize encapsulation. Once single cell containing droplets are generated, they are transferred to a DMF chip via a capillary where they are merged with droplets containing IL and cultured at 30 °C. The DMF chip, in addition to permitting cell culture and reagent (ionic liquid/salt) addition, also allows recovery of individual droplets for off-chip analysis such as further culturing and measurement of ethanol production. The D2D chip was used to evaluate the effect of IL/salt type (four types: NaOAc, NaCl, [C2mim] [OAc], [C2mim] [Cl]) and concentration (four concentrations: 0, 37.5, 75, 150 mM) on the growth kinetics and ethanol production of yeast and as expected, increasing IL concentration led to lower biomass and ethanol production. Specifically, [C2mim] [OAc] had inhibitory effects on yeast growth at concentrations 75 and 150 mM and significantly reduced their ethanol production compared to cells grown in other ILs/salts. The growth curve trends obtained by D2D matched conventional yeast culturing in microtiter wells, validating the D2D platform. We believe that our approach represents a generic platform for multi-step biochemical assays such as drug screening, digital PCR, enzyme assays, immunoassays and cell-based assays.

HCN1 channels in cerebellar Purkinje cells promote late stages of learning and constrain synaptic inhibition

PubMed Central

Rinaldi, Arianna; Defterali, Cagla; Mialot, Antoine; Garden, Derek L F; Beraneck, Mathieu; Nolan, Matthew F

2013-01-01

Neural computations rely on ion channels that modify neuronal responses to synaptic inputs. While single cell recordings suggest diverse and neurone type-specific computational functions for HCN1 channels, their behavioural roles in any single neurone type are not clear. Using a battery of behavioural assays, including analysis of motor learning in vestibulo-ocular reflex and rotarod tests, we find that deletion of HCN1 channels from cerebellar Purkinje cells selectively impairs late stages of motor learning. Because deletion of HCN1 modifies only a subset of behaviours involving Purkinje cells, we asked whether the channel also has functional specificity at a cellular level. We find that HCN1 channels in cerebellar Purkinje cells reduce the duration of inhibitory synaptic responses but, in the absence of membrane hyperpolarization, do not affect responses to excitatory inputs. Our results indicate that manipulation of subthreshold computation in a single neurone type causes specific modifications to behaviour. PMID:24000178

Using optical tweezers to examine the chemotactic force to a single inflammatory cell--eosinophil stimulated by chemoattractants prepared from Toxocara Canis larvae

NASA Astrophysics Data System (ADS)

Shih, Po-Chen; Su, Yi-Jr; Chen, Ke-Min; Jen, Lin-Ni; Liu, Cheng-tzu; Hsu, Long

2005-08-01

Granulocytes are a group of white blood cells belonging to the innate immune system in human and in murine in which eosinophils play an important role in worm infection-induced inflammation. The migration of these cells is well characterized and has been separated into four steps: rolling, adhesion, transendothelial migration, and chemotaxis, however, the physical characteristics of the chemotactic force to eosinophils from worm component remain largely unknown. Note that optical tweezers are featured in the manipulation of a single cell and the measurement of biological forces. Therefore, we propose to use optical tweezers to examine the chemotactic force to a eosinophil from a T. canis lavae preparation in terms of distance during the migration of eosinophil.

Near-infrared Raman spectroscopy of single optically trapped biological cells

NASA Astrophysics Data System (ADS)

Xie, Changan; Dinno, Mumtaz A.; Li, Yong-Qing

2002-02-01

We report on the development and testing of a compact laser tweezers Raman spectroscopy (LTRS) system. The system combines optical trapping and near-infrared Raman spectroscopy for manipulation and identification of single biological cells in solution. A low-power diode laser at 785 nm was used for both trapping and excitation for Raman spectroscopy of the suspended microscopic particles. The design of the LTRS system provides high sensitivity and permits real-time spectroscopic measurements of the biological sample. The system was calibrated by use of polystyrene microbeads and tested on living blood cells and on both living and dead yeast cells. As expected, different images and Raman spectra were observed for the different cells. The LTRS system may provide a valuable tool for the study of fundamental cellular processes and the diagnosis of cellular disorders.

The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation.

PubMed

Collins, David J; Neild, Adrian; deMello, Andrew; Liu, Ai-Qun; Ai, Ye

2015-09-07

There is a recognized and growing need for rapid and efficient cell assays, where the size of microfluidic devices lend themselves to the manipulation of cellular populations down to the single cell level. An exceptional way to analyze cells independently is to encapsulate them within aqueous droplets surrounded by an immiscible fluid, so that reagents and reaction products are contained within a controlled microenvironment. Most cell encapsulation work has focused on the development and use of passive methods, where droplets are produced continuously at high rates by pumping fluids from external pressure-driven reservoirs through defined microfluidic geometries. With limited exceptions, the number of cells encapsulated per droplet in these systems is dictated by Poisson statistics, reducing the proportion of droplets that contain the desired number of cells and thus the effective rate at which single cells can be encapsulated. Nevertheless, a number of recently developed actively-controlled droplet production methods present an alternative route to the production of droplets at similar rates and with the potential to improve the efficiency of single-cell encapsulation. In this critical review, we examine both passive and active methods for droplet production and explore how these can be used to deterministically and non-deterministically encapsulate cells.

Cell separation and electrofusion in space

NASA Technical Reports Server (NTRS)

Morrison, D. R.; Hofmann, G. A.

1990-01-01

In microgravity, free-fluid electrophoretic methods for separating living cells and proteins are improved significantly by the absence of gravity-driven phenomena. Cell fusion, culture, and other bioprocessing steps are being investigated to understand the limits of earth-based processing. A multistep space bioprocess is described that includes electrophoretic separation of human target cells, single-cell manipulations using receptor-specific antibodies, electrofusion to produce immortal hybridomas, gentle suspension culture, and monoclonal antibody recovery using continuous-flow electrophoresis or recirculating isoelectric focusing. Improvements in several key steps already have been demonstrated by space experiments, and others will be studied on Space Station Freedom.

Lab-on-a-chip technologies for proteomic analysis from isolated cells

PubMed Central

Sedgwick, H.; Caron, F.; Monaghan, P.B.; Kolch, W.; Cooper, J.M.

2008-01-01

Lab-on-a-chip systems offer a versatile environment in which low numbers of cells and molecules can be manipulated, captured, detected and analysed. We describe here a microfluidic device that allows the isolation, electroporation and lysis of single cells. A431 human epithelial carcinoma cells, expressing a green fluorescent protein-labelled actin, were trapped by dielectrophoresis within an integrated lab-on-a-chip device containing saw-tooth microelectrodes. Using these same trapping electrodes, on-chip electroporation was performed, resulting in cell lysis. Protein release was monitored by confocal fluorescence microscopy. PMID:18534931

Genetic Engineering of Optical Properties of Biomaterials

NASA Astrophysics Data System (ADS)

Gourley, Paul; Naviaux, Robert; Yaffe, Michael

2008-03-01

Baker's yeast cells are easily cultured and can be manipulated genetically to produce large numbers of bioparticles (cells and mitochondria) with controllable size and optical properties. We have recently employed nanolaser spectroscopy to study the refractive index of individual cells and isolated mitochondria from two mutant strains. Results show that biomolecular changes induced by mutation can produce bioparticles with radical changes in refractive index. Wild-type mitochondria exhibit a distribution with a well-defined mean and small variance. In striking contrast, mitochondria from one mutant strain produced a histogram that is highly collapsed with a ten-fold decrease in the mean and standard deviation. In a second mutant strain we observed an opposite effect with the mean nearly unchanged but the variance increased nearly a thousand-fold. Both histograms could be self-consistently modeled with a single, log-normal distribution. The strains were further examined by 2-dimensional gel electrophoresis to measure changes in protein composition. All of these data show that genetic manipulation of cells represents a new approach to engineering optical properties of bioparticles.

Local x-ray structure analysis of optically manipulated biological micro-objects

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cojoc, Dan; Ferrari, Enrico; Santucci, Silvia C.

2010-12-13

X-ray diffraction using micro- and nanofocused beams is well suited for nanostructure analysis at different sites of a biological micro-object. To conduct in vitro studies without mechanical contact, we developed object manipulation by optical tweezers in a microfluidic cell. Here we report x-ray microdiffraction analysis of a micro-object optically trapped in three dimensions. We revealed the nanostructure of a single starch granule at different points and investigated local radiation damage induced by repeated x-ray exposures at the same position, demonstrating high stability and full control of the granule orientation by multiple optical traps.

A rapid single-tube protocol for HAV detection by nested real-time PCR.

PubMed

Hu, Yuan; Arsov, Ivica

2014-09-01

Infections by food-borne viruses such as hepatitis A virus (HAV) and norovirus are significant public health concerns worldwide. Since food-borne viruses are rarely confirmed through direct isolation from contaminated samples, highly sensitive molecular techniques remain the methods of choice for the detection of viral genetic material. Our group has previously developed a specific nested real-time PCR (NRT-PCR) assay for HAV detection that improved overall sensitivity. Furthermore in this study, we have developed a single-tube NRT-PCR approach for HAV detection in food samples that reduces the likelihood of cross contamination between tubes during sample manipulation. HAV RNA was isolated from HAV-spiked food samples and HAV-infected cell cultures. All reactions following HAV RNA isolation, including conventional reverse transcriptase PCR, nested-PCR, and RT-PCR were performed in a single tube. Our results demonstrated that all the samples tested positive by RT-PCR and nested-PCR were also positive by a single-tube NRT-PCR. The detection limits observed for HAV-infected cell cultures and HAV-spiked green onions were 0.1 and 1 PFU, respectively. This novel method retained the specificity and robustness of the original NRT-PCR method, while greatly reducing sample manipulation, turnaround time, and the risk of carry-over contamination. Single-tube NRT-PCR thus represents a promising new tool that can potentially facilitate the detection of HAV in foods thereby improving food safety and public health.

In-vivo RGB marking and multicolour single-cell tracking in the adult brain

PubMed Central

Gomez-Nicola, Diego; Riecken, Kristoffer; Fehse, Boris; Perry, V. Hugh

2014-01-01

In neuroscience it is a technical challenge to identify and follow the temporal and spatial distribution of cells as they differentiate. We hypothesised that RGB marking, the tagging of individual cells with unique hues resulting from simultaneous expression of the three basic colours red, green and blue, provides a convenient toolbox for the study of the CNS anatomy at the single-cell level. Using γ-retroviral and lentiviral vector sets we describe for the first time the in-vivo multicolour RGB marking of neurons in the adult brain. RGB marking also enabled us to track the spatial and temporal fate of neural stem cells in the adult brain. The application of different viral envelopes and promoters provided a useful approach to track the generation of neurons vs. glial cells at the neurogenic niche, allowing the identification of the prominent generation of new astrocytes to the striatum. Multicolour RGB marking could serve as a universal and reproducible method to study and manipulate the CNS at the single-cell level, in both health and disease. PMID:25531807

Variation is function: Are single cell differences functionally important?: Testing the hypothesis that single cell variation is required for aggregate function.

PubMed

Dueck, Hannah; Eberwine, James; Kim, Junhyong

2016-02-01

There is a growing appreciation of the extent of transcriptome variation across individual cells of the same cell type. While expression variation may be a byproduct of, for example, dynamic or homeostatic processes, here we consider whether single-cell molecular variation per se might be crucial for population-level function. Under this hypothesis, molecular variation indicates a diversity of hidden functional capacities within an ensemble of identical cells, and this functional diversity facilitates collective behavior that would be inaccessible to a homogenous population. In reviewing this topic, we explore possible functions that might be carried by a heterogeneous ensemble of cells; however, this question has proven difficult to test, both because methods to manipulate molecular variation are limited and because it is complicated to define, and measure, population-level function. We consider several possible methods to further pursue the hypothesis that variation is function through the use of comparative analysis and novel experimental techniques. © 2015 The Authors. BioEssays published by WILEY Periodicals, Inc.

Non-invasive optoacoustic probing of the density and stiffness of single biological cells

NASA Astrophysics Data System (ADS)

Dehoux, T.; Audoin, B.

2012-12-01

Recently, the coherent generation of GHz acoustic waves using ultrashort laser pulses has demonstrated the ability to probe the sound velocity in vegetal cells and in cell-mimicking soft micro-objects with micrometer resolution, opening tremendous potentialities for single-cell biology. However, manipulating biological media in physiological conditions is often a technical challenge when using a laser-based setup. In this article, we present a new opto-acoustic bio-transducer composed of a thin metal film sputtered on a transparent heat sink that allows reducing importantly the laser-induced cellular stresses, and offers a wide variety of optical configurations. In particular, by exploiting the acoustic reflection coefficient at the sample-transducer interface and the photoacoustic interaction inside the transparent sample, the density and compressibility of the sample can be probed simultaneously. Using an ad hoc signal analysis based on Hilbert and wavelet transforms, these quantities are measured accurately for a reference fluid. Similar analysis performed in a single vegetal cell also suggests high sensitivity to the state of the transducer-cell interface, and notably to the presence of the plasma membrane that encloses the cell vacuole.

Host-regulated Hepatitis B Virus Capsid Assembly in a Mammalian Cell-free System.

PubMed

Liu, Kuancheng; Hu, Jianming

2018-04-20

The hepatitis B virus (HBV) is an important global human pathogen and represents a major cause of hepatitis, liver cirrhosis and liver cancer. The HBV capsid is composed of multiple copies of a single viral protein, the capsid or core protein (HBc), plays multiple roles in the viral life cycle, and has emerged recently as a major target for developing antiviral therapies against HBV infection. Although several systems have been developed to study HBV capsid assembly, including heterologous overexpression systems like bacteria and insect cells, in vitro assembly using purified protein, and mammalian cell culture systems, the requirement for non-physiological concentrations of HBc and salts and the difficulty in manipulating host regulators of assembly presents major limitations for detailed studies on capsid assembly under physiologically relevant conditions. We have recently developed a mammalian cell-free system based on the rabbit reticulocyte lysate (RRL), in which HBc is expressed at physiological concentrations and assembles into capsids under near-physiological conditions. This system has already revealed HBc assembly requirements that are not anticipated based on previous assembly systems. Furthermore, capsid assembly in this system is regulated by endogenous host factors that can be readily manipulated. Here we present a detailed protocol for this cell-free capsid assembly system, including an illustration on how to manipulate host factors that regulate assembly.

Atomic force microscopy for cellular level manipulation: imaging intracellular structures and DNA delivery through a membrane hole.

PubMed

Afrin, Rehana; Zohora, Umme Salma; Uehara, Hironori; Watanabe-Nakayama, Takahiro; Ikai, Atsushi

2009-01-01

The atomic force microscope (AFM) is a versatile tool for imaging, force measurement and manipulation of proteins, DNA, and living cells basically at the single molecular level. In the cellular level manipulation, extraction, and identification of mRNA's from defined loci of a cell, insertion of plasmid DNA and pulling of membrane proteins, for example, have been reported. In this study, AFM was used to create holes at defined loci on the cell membrane for the investigation of viability of the cells after hole creation, visualization of intracellular structure through the hole and for targeted gene delivery into living cells. To create large holes with an approximate diameter of 5-10 microm, a phospholipase A(2) coated bead was added to the AFM cantilever and the bead was allowed to touch the cell surface for approximately 5-10 min. The evidence of hole creation was obtained mainly from fluorescent image of Vybrant DiO labeled cell before and after the contact with the bead and the AFM imaging of the contact area. In parallel, cells with a hole were imaged by AFM to reveal intracellular structures such as filamentous structures presumably actin fibers and mitochondria which were identified with fluorescent labeling with rhodamine 123. Targeted gene delivery was also attempted by inserting an AFM probe that was coated with the Monster Green Fluorescent Protein phMGFP Vector for transfection of the cell. Following targeted transfection, the gene expression of green fluorescent protein (GFP) was observed and confirmed by the fluorescence microscope. Copyright (c) 2009 John Wiley & Sons, Ltd.

Live cell isolation by laser microdissection with gravity transfer

NASA Astrophysics Data System (ADS)

Podgorny, Oleg V.

2013-05-01

Laser microdissection by pulsing ultraviolet laser allows the isolation and recultivation of live cells based on morphological features or/and fluorescent labelling from adherent cell cultures. Previous investigations described only the use of the laser microdissection and pressure catapulting (LMPC) for live cell isolation. But LMPC requires complex manipulations and some skill. Furthermore, single-cell cloning using laser microdissection has not yet been demonstrated. The first evidence of successful application of laser microdissection with gravity transfer (LMDGT) for capturing and recultivation of live cells is presented. A new strategy for LMDGT is presented because of the failure to reproduce the manufacturer's protocol. Using the new strategy, successful capturing and recultivation of circle-shaped samples from confluent monolayer of HeLa cells was demonstrated. It was found that LMDGT is easier than LMPC because it doesn't require personal participation of investigator in transferring of isolated samples to final culture dishes. Moreover, for the first time, the generation of clonal colonies from single live cells isolated by laser microdissection was demonstrated. Data obtained in this study confirm that LMDGT is a reliable and high-yield method allowing isolation and expansion of both cell clusters and single cells from adherent cell cultures.

Three-dimensional ultrasonic trapping of micro-particles in water with a simple and compact two-element transducer

NASA Astrophysics Data System (ADS)

Franklin, A.; Marzo, A.; Malkin, R.; Drinkwater, B. W.

2017-08-01

We report a simple and compact piezoelectric transducer capable of stably trapping single and multiple micro-particles in water. A 3D-printed Fresnel lens is bonded to a two-element kerfless piezoceramic disk and actuated in a split-piston mode to produce an acoustic radiation force trap that is stable in three-dimensions. Polystyrene micro-particles in the Rayleigh regime (radius λ/14 to λ/7) are trapped at the focus of the lens (F# = 0.4) and manipulated in two-dimensions on an acoustically transparent membrane with a peak trap stiffness of 0.43 mN/m. Clusters of Rayleigh particles are also trapped and manipulated in three-dimensions, suspended in water against gravity. This transducer represents a significant simplification over previous acoustic devices used for micro-particle manipulation in liquids as it operates at relatively low frequency (688 kHz) and only requires a single electrical drive signal. This simplified device has potential for widespread use in applications such as micro-scale manufacturing and handling of cells or drug capsules in biomedical assays.

Microparticles controllable accumulation, arrangement, and spatial shaping performed by tapered-fiber-based laser-induced convection flow.

PubMed

Zhang, Yu; Lei, Jiaojie; Zhang, Yaxun; Liu, Zhihai; Zhang, Jianzhong; Yang, Xinghua; Yang, Jun; Yuan, Libo

2017-10-30

The ability to arrange cells and/or microparticles into the desired pattern is critical in biological, chemical, and metamaterial studies and other applications. Researchers have developed a variety of patterning techniques, which either have a limited capacity to simultaneously trap massive particles or lack the spatial resolution necessary to manipulate individual particle. Several approaches have been proposed that combine both high spatial selectivity and high throughput simultaneously. However, those methods are complex and difficult to fabricate. In this article, we propose and demonstrate a simple method that combines the laser-induced convection flow and fiber-based optical trapping methods to perform both regular and special spatial shaping arrangement. Essentially, we combine a light field with a large optical intensity gradient distribution and a thermal field with a large temperature gradient distribution to perform the microparticles shaping arrangement. The tapered-fiber-based laser-induced convection flow provides not only the batch manipulation of massive particles, but also the finer manipulation of special one or several particles, which break out the limit of single-fiber-based massive/individual particles photothermal manipulation. The combination technique allows for microparticles quick accumulation, single-layer and multilayer arrangement; special spatial shaping arrangement/adjustment, and microparticles sorting.

Single Particle Differentiation through 2D Optical Fiber Trapping and Back-Scattered Signal Statistical Analysis: An Exploratory Approach

PubMed Central

Ribeiro, Rita S. R.; Cunha, João P. S.; Rosa, Carla C.; Jorge, Pedro A. S.

2018-01-01

Recent trends on microbiology point out the urge to develop optical micro-tools with multifunctionalities such as simultaneous manipulation and sensing. Considering that miniaturization has been recognized as one of the most important paradigms of emerging sensing biotechnologies, optical fiber tools, including Optical Fiber Tweezers (OFTs), are suitable candidates for developing multifunctional small sensors for Medicine and Biology. OFTs are flexible and versatile optotools based on fibers with one extremity patterned to form a micro-lens. These are able to focus laser beams and exert forces onto microparticles strong enough (piconewtons) to trap and manipulate them. In this paper, through an exploratory analysis of a 45 features set, including time and frequency-domain parameters of the back-scattered signal of particles trapped by a polymeric lens, we created a novel single feature able to differentiate synthetic particles (PMMA and Polystyrene) from living yeasts cells. This single statistical feature can be useful for the development of label-free hybrid optical fiber sensors with applications in infectious diseases detection or cells sorting. It can also contribute, by revealing the most significant information that can be extracted from the scattered signal, to the development of a simpler method for particles characterization (in terms of composition, heterogeneity degree) than existent technologies. PMID:29495502

Single Particle Differentiation through 2D Optical Fiber Trapping and Back-Scattered Signal Statistical Analysis: An Exploratory Approach.

PubMed

Paiva, Joana S; Ribeiro, Rita S R; Cunha, João P S; Rosa, Carla C; Jorge, Pedro A S

2018-02-27

Recent trends on microbiology point out the urge to develop optical micro-tools with multifunctionalities such as simultaneous manipulation and sensing. Considering that miniaturization has been recognized as one of the most important paradigms of emerging sensing biotechnologies, optical fiber tools, including Optical Fiber Tweezers (OFTs), are suitable candidates for developing multifunctional small sensors for Medicine and Biology. OFTs are flexible and versatile optotools based on fibers with one extremity patterned to form a micro-lens. These are able to focus laser beams and exert forces onto microparticles strong enough (piconewtons) to trap and manipulate them. In this paper, through an exploratory analysis of a 45 features set, including time and frequency-domain parameters of the back-scattered signal of particles trapped by a polymeric lens, we created a novel single feature able to differentiate synthetic particles (PMMA and Polystyrene) from living yeasts cells. This single statistical feature can be useful for the development of label-free hybrid optical fiber sensors with applications in infectious diseases detection or cells sorting. It can also contribute, by revealing the most significant information that can be extracted from the scattered signal, to the development of a simpler method for particles characterization (in terms of composition, heterogeneity degree) than existent technologies.

Single-cell intracellular nano-pH probes†

PubMed Central

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

A molecular scheme for improved characterization of human embryonic stem cell lines

PubMed Central

Josephson, Richard; Sykes, Gregory; Liu, Ying; Ording, Carol; Xu, Weining; Zeng, Xianmin; Shin, Soojung; Loring, Jeanne; Maitra, Anirban; Rao, Mahendra S; Auerbach, Jonathan M

2006-01-01

Background Human embryonic stem cells (hESC) offer a renewable source of a wide range of cell types for use in research and cell-based therapies to treat disease. Inspection of protein markers provides important information about the current state of the cells and data for subsequent manipulations. However, hESC must be routinely analyzed at the genomic level to guard against deleterious changes during extensive propagation, expansion, and manipulation in vitro. Results We found that short tandem repeat (STR) analysis, human leukocyte antigen (HLA) typing, single nucleotide polymorphism (SNP) genomic analysis, mitochondrial DNA sequencing, and gene expression analysis by microarray can be used to fully describe any hESC culture in terms of its identity, stability, and undifferentiated state. Conclusion Here we describe, using molecular biology alone, a comprehensive characterization of 17 different hESC lines. The use of amplified nucleic acids means that for the first time full characterization of hESC lines can be performed with little time investment and a minimum of material. The information thus gained will facilitate comparison of lines and replication of results between laboratories. PMID:16919167

A single frame: imaging live cells twenty-five years ago.

PubMed

Fink, Rachel

2011-07-01

In the mid-1980s live-cell imaging was changed by the introduction of video techniques, allowing new ways to collect and store data. The increased resolution obtained by manipulating video signals, the ability to use time-lapse videocassette recorders to study events that happen over long time intervals, and the introduction of fluorescent probes and sensitive video cameras opened research avenues previously unavailable. The author gives a personal account of this evolution, focusing on cell migration studies at the Marine Biological Laboratory 25 years ago. Copyright © 2011 Wiley-Liss, Inc.

A Molecular Analysis of Training Multiple versus Single Manipulations to Establish a Generalized Manipulative Imitation Repertoire

ERIC Educational Resources Information Center

Hartley, Breanne K.

2009-01-01

This study evaluates the necessity of training multiple versus single manipulative-imitations per object in order to establish generalized manipulative-imitation. Training took place in Croyden Avenue School's Early Childhood Developmental Delay preschool classroom in Kalamazoo, MI. Two groups of 3 children each were trained to imitate in order to…

Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance.

PubMed

Ito, Kyoko; Turcotte, Raphaël; Cui, Jinhua; Zimmerman, Samuel E; Pinho, Sandra; Mizoguchi, Toshihide; Arai, Fumio; Runnels, Judith M; Alt, Clemens; Teruya-Feldstein, Julie; Mar, Jessica C; Singh, Rajat; Suda, Toshio; Lin, Charles P; Frenette, Paul S; Ito, Keisuke

2016-12-02

A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms of HSC division balance, however, are not yet defined. Here we demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2 + HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction of mitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2 + HSCs. Activation of the PPAR (peroxisome proliferator-activated receptor)-fatty acid oxidation pathway promotes expansion of Tie2 + HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2 + HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways. Copyright © 2016, American Association for the Advancement of Science.

Self-renewal of a purified Tie2+ hematopoietic stem cell population relies on mitochondrial clearance

PubMed Central

Ito, Kyoko; Turcotte, Raphaël; Cui, Jinhua; Zimmerman, Samuel E.; Pinho, Sandra; Mizoguchi, Toshihide; Arai, Fumio; Runnels, Judith M.; Alt, Clemens; Teruya-Feldstein, Julie; Mar, Jessica C.; Singh, Rajat; Suda, Toshio; Lin, Charles P.; Frenette, Paul S.; Ito, Keisuke

2016-01-01

A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms of HSC division balance, however, are not yet defined. Here we demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2+ HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction of mitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2+ HSCs. Activation of the PPAR (peroxisome proliferator–activated receptor)–fatty acid oxidation pathway promotes expansion of Tie2+ HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2+ HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways. PMID:27738012

Higher-order micro-fiber modes for Escherichia coli manipulation using a tapered seven-core fiber

PubMed Central

Rong, Qiangzhou; Zhou, Yi; Yin, Xunli; Shao, Zhihua; Qiao, Xueguang

2017-01-01

Optical manipulation using optical micro- and nano-fibers has shown potential for controlling bacterial activities such as E. coli trapping, propelling, and binding. Most of these manipulations have been performed using the propagation of the fundamental mode through the fiber. However, along the maximum mode-intensity axis, the higher-order modes have longer evanescent field extensions and larger field amplitudes at the fiber waist than the fundamental mode, opening up new possibilities for manipulating E. coli bacteria. In this work, a compact seven-core fiber (SCF)-based micro-fiber/optical tweezers was demonstrated for trapping, propelling, and rotating E. coli bacteria using the excitation of higher-order modes. The diameter of the SCF taper was 4 µm at the taper waist, which was much larger than that of previous nano-fiber tweezers. The laser wavelength was tunable from 1500 nm to 1600 nm, simultaneously causing photophoretic force, gradient force, and scattering force. This work provides a new opportunity for better understanding optical manipulation using higher-order modes at the single-cell level. PMID:28966849

Higher-order micro-fiber modes for Escherichia coli manipulation using a tapered seven-core fiber.

PubMed

Rong, Qiangzhou; Zhou, Yi; Yin, Xunli; Shao, Zhihua; Qiao, Xueguang

2017-09-01

Optical manipulation using optical micro- and nano-fibers has shown potential for controlling bacterial activities such as E. coli trapping, propelling, and binding. Most of these manipulations have been performed using the propagation of the fundamental mode through the fiber. However, along the maximum mode-intensity axis, the higher-order modes have longer evanescent field extensions and larger field amplitudes at the fiber waist than the fundamental mode, opening up new possibilities for manipulating E. coli bacteria. In this work, a compact seven-core fiber (SCF)-based micro-fiber/optical tweezers was demonstrated for trapping, propelling, and rotating E. coli bacteria using the excitation of higher-order modes. The diameter of the SCF taper was 4 µm at the taper waist, which was much larger than that of previous nano-fiber tweezers. The laser wavelength was tunable from 1500 nm to 1600 nm, simultaneously causing photophoretic force, gradient force, and scattering force. This work provides a new opportunity for better understanding optical manipulation using higher-order modes at the single-cell level.

Cell Membrane Deformation Induced by a Fibronectin-Coated Polystyrene Microbead in a 200-MHz Acoustic Trap

PubMed Central

Hwang, Jae Youn; Lee, Changyang; Lam, Kwok Ho; Kim, Hyung Ham; Lee, Jungwoo; Shung, K. Kirk

2014-01-01

The measurement of cell mechanics is crucial for a better understanding of cellular responses during the progression of certain diseases and for the identification of the cell’s nature. Many techniques using optical tweezers, atomic force microscopy, and micro-pipettes have been developed to probe and manipulate cells in the spatial domain. In particular, we recently proposed a two-dimensional acoustic trapping method as an alternative technique for small particle manipulation. Although the proposed method may have advantages over optical tweezers, its applications to cellular mechanics have not yet been vigorously investigated. This study represents an initial attempt to use acoustic tweezers as a tool in the field of cellular mechanics in which cancer cell membrane deformability is studied. A press-focused 193-MHz single-element lithium niobate (LiNbO3) transducer was designed and fabricated to trap a 5-µm polystyrene microbead near the ultrasound beam focus. The microbeads were coated with fibronectin, and trapped before being attached to the surface of a human breast cancer cell (MCF-7). The cell membrane was then stretched by remotely pulling a cell-attached microbead with the acoustic trap. The maximum cell membrane stretched lengths were measured to be 0.15, 0.54, and 1.41 µm at input voltages to the transducer of 6.3, 9.5, and 12.6 Vpp, respectively. The stretched length was found to increase nonlinearly as a function of the voltage input. No significant cytotoxicity was observed to result from the bead or the trapping force on the cell during or after the deformation procedure. Hence, the results convincingly demonstrated the possible application of the acoustic trapping technique as a tool for cell manipulation. PMID:24569245

Vision-guided micromanipulation system for biomedical application

NASA Astrophysics Data System (ADS)

Shim, Jae-Hong; Cho, Sung-Yong; Cha, Dong-Hyuk

2004-10-01

In these days, various researches for biomedical application of robots have been carried out. Particularly, robotic manipulation of the biological cells has been studied by many researchers. Usually, most of the biological cell's shape is sphere. Commercial biological manipulation systems have been utilized the 2-Dimensional images through the optical microscopes only. Moreover, manipulation of the biological cells mainly depends on the subjective viewpoint of an operator. Due to these reasons, there exist lots of problems such as slippery and destruction of the cell membrane and damage of the pipette tip etc. In order to overcome the problems, we have proposed a vision-guided biological cell manipulation system. The newly proposed manipulation system makes use of vision and graphic techniques. Through the proposed procedures, an operator can inject the biological cell scientifically and objectively. Also, the proposed manipulation system can measure the contact force occurred at injection of a biological cell. It can be transmitted a measured force to the operator by the proposed haptic device. Consequently, the proposed manipulation system could safely handle the biological cells without any damage. This paper presents the introduction of our vision-guided manipulation techniques and the concept of the contact force sensing. Through a series of experiments the proposed vision-guided manipulation system shows the possibility of application for precision manipulation of the biological cell such as DNA.

Electrokinetic gated injection-based microfluidic system for quantitative analysis of hydrogen peroxide in individual HepG2 cells.

PubMed

Zhang, Xinyuan; Li, Qingling; Chen, Zhenzhen; Li, Hongmin; Xu, Kehua; Zhang, Lisheng; Tang, Bo

2011-03-21

A microfluidic system to determine hydrogen peroxide (H(2)O(2)) in individual HepG2 cells based on the electrokinetic gated injection was developed for the first time. A home-synthesized fluorescent probe, bis(p-methylbenzenesulfonate)dichlorofluorescein (FS), was employed to label intracellular H(2)O(2) in the intact cells. On a simple cross microchip, multiple single-cell operations, including single cell injection, cytolysis, electrophoresis separation and detection of H(2)O(2), were automatically carried out within 60 s using the electrokinetic gated injection and laser-induced fluorescence detection (LIFD). The performance of the method was evaluated under the optimal conditions. The linear calibration curve was over a range of 4.39-610 amol (R(2)=0.9994). The detection limit was 0.55 amol or 9.0×10(-10) M (S/N=3). The relative standard deviations (RSDs, n=6) of migration time and peak area were 1.4% and 4.8%, respectively. With the use of this method, the average content of H(2)O(2) in single HepG2 cells was found to be 16.09±9.84 amol (n=15). Separation efficiencies in excess of 17,000 theoretical plates for the cells were achieved. These results demonstrated that the efficient integration and automation of these single-cell operations enabled the sensitive, reproducible, and quantitative examination of intracellular H(2)O(2) at single-cell level. Owing to the advantages of simple microchip structure, controllable single-cell manipulation and ease in building, this platform provides a universal way to automatically determine other intracellular constituents within single cells. This journal is © The Royal Society of Chemistry 2011

Optical micromanipulation of active cells with minimal perturbations: direct and indirect pushing.

PubMed

Wang, Chenlu; Chowdhury, Sagar; Gupta, Satyandra K; Losert, Wolfgang

2013-04-01

The challenge to wide application of optical tweezers in biological micromanipulation is the photodamage caused by high-intensity laser exposure to the manipulated living systems. While direct exposure to infrared lasers is less likely to kill cells, it can affect cell behavior and signaling. Pushing cells with optically trapped objects has been introduced as a less invasive alternative, but the technique includes some exposure of the biological object to parts of the optical tweezer beam. To keep the cells farther away from the laser, we introduce an indirect pushing-based technique for noninvasive manipulation of sensitive cells. We compare how cells respond to three manipulation approaches: direct manipulation, pushing, and indirect pushing. We find that indirect manipulation techniques lessen the impact of manipulation on cell behavior. Cell survival increases, as does the ability of cells to maintain shape and wiggle. Our experiments also demonstrate that indirect pushing allows cell-cell contacts to be formed in a controllable way, while retaining the ability of cells to change shape and move.

Nanolaser spectroscopy and micro-optical resonators for detecting, analyzing, and manipulating bioparticles

DOEpatents

Gourley, Paul L

2012-06-26

This invention provides a new method for rapidly analyzing single bioparticles to assess their material condition and state of health. The method is enabled by use of a resonant cavity apparatus to measure an optical property related to the bioparticle size and refractive index. Measuring the refractive index is useful for determining material properties of the bioparticle. The material properties depend on the biomolecular composition of the bioparticle. The biomolecular composition is, in turn, dependent on the state of health of the bioparticle. Thus, measured optical properties can be used to differentiate normal (healthy) and abnormal (diseased) states of bioparticles derived from cells or tissues. The method is illustrated with data obtained from a resonator with a gain medium. The invention also provides new methods for making multiple measurements in a single device and detecting, analyzing, and manipulating bioparticles that are much smaller than the wavelength of light.

The structure and function of cell membranes studied by atomic force microscopy.

PubMed

Shi, Yan; Cai, Mingjun; Zhou, Lulu; Wang, Hongda

2018-01-01

The cell membrane, involved in almost all communications of cells and surrounding matrix, is one of the most complicated components of cells. Lack of suitable methods for the detection of cell membranes in vivo has sparked debates on the biochemical composition and structure of cell membranes over half a century. The development of single molecule techniques, such as AFM, SMFS, and TREC, provides a versatile platform for imaging and manipulating cell membranes in biological relevant environments. Here, we discuss the latest developments in AFM and the progress made in cell membrane research. In particular, we highlight novel structure models and dynamic processes, including the mechanical properties of the cell membranes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Raman-Activated Droplet Sorting (RADS) for Label-Free High-Throughput Screening of Microalgal Single-Cells.

PubMed

Wang, Xixian; Ren, Lihui; Su, Yetian; Ji, Yuetong; Liu, Yaoping; Li, Chunyu; Li, Xunrong; Zhang, Yi; Wang, Wei; Hu, Qiang; Han, Danxiang; Xu, Jian; Ma, Bo

2017-11-21

Raman-activated cell sorting (RACS) has attracted increasing interest, yet throughput remains one major factor limiting its broader application. Here we present an integrated Raman-activated droplet sorting (RADS) microfluidic system for functional screening of live cells in a label-free and high-throughput manner, by employing AXT-synthetic industrial microalga Haematococcus pluvialis (H. pluvialis) as a model. Raman microspectroscopy analysis of individual cells is carried out prior to their microdroplet encapsulation, which is then directly coupled to DEP-based droplet sorting. To validate the system, H. pluvialis cells containing different levels of AXT were mixed and underwent RADS. Those AXT-hyperproducing cells were sorted with an accuracy of 98.3%, an enrichment ratio of eight folds, and a throughput of ∼260 cells/min. Of the RADS-sorted cells, 92.7% remained alive and able to proliferate, which is equivalent to the unsorted cells. Thus, the RADS achieves a much higher throughput than existing RACS systems, preserves the vitality of cells, and facilitates seamless coupling with downstream manipulations such as single-cell sequencing and cultivation.

Investigation of Biophysical Mechanisms in Gold Nanoparticle Mediated Laser Manipulation of Cells Using a Multimodal Holographic and Fluorescence Imaging Setup

PubMed Central

Rakoski, Mirko S.; Heinemann, Dag; Schomaker, Markus; Ripken, Tammo; Meyer, Heiko

2015-01-01

Laser based cell manipulation has proven to be a versatile tool in biomedical applications. In this context, combining weakly focused laser pulses and nanostructures, e.g. gold nanoparticles, promises to be useful for high throughput cell manipulation, such as transfection and photothermal therapy. Interactions between laser pulses and gold nanoparticles are well understood. However, it is still necessary to study cell behavior in gold nanoparticle mediated laser manipulation. While parameters like cell viability or perforation efficiency are commonly addressed, the influence of the manipulation process on other essential cell parameters is not sufficiently investigated yet. Thus, we set out to study four relevant cell properties: cell volume and area, ion exchange and cytoskeleton structure after gold nanoparticle based laser manipulation. For this, we designed a multimodal imaging and manipulation setup. 200 nm gold nanoparticles were attached unspecifically to canine cells and irradiated by weakly focused 850 ps laser pulses. Volume and area change in the first minute post laser manipulation was monitored using digital holography. Calcium imaging and cells expressing a marker for filamentous actin (F-actin) served to analyze the ion exchange and the cytoskeleton, respectively. High radiant exposures led to cells exhibiting a tendency to shrink in volume and area, possibly due to outflow of cytoplasm. An intracellular raise in calcium was observed and accompanied by an intercellular calcium wave. This multimodal approach enabled for the first time a comprehensive analysis of the cell behavior in gold nanoparticle mediated cell manipulation. Additionally, this work can pave the way for a better understanding and the evaluation of new applications in the context of cell transfection or photothermal therapy. PMID:25909631

Transgenic manipulation of a single polyamine in poplar cells affects the accumulation of all amino acids

Treesearch

Sridev Mohapatra; Rakesh Minocha; Stephanie Long; Subhash C. Minocha

2010-01-01

The polyamine metabolic pathway is intricately connected to metabolism of several amino acids. While ornithine and arginine are direct precursors of putrescine, they themselves are synthesized from glutamate in multiple steps involving several enzymes. Additionally, glutamate is an amino group donor for several other amino acids and acts as a substrate for biosynthesis...

Characterization of femtosecond-laser pulse induced cell membrane nanosurgical attachment.

PubMed

Katchinskiy, Nir; Godbout, Roseline; Elezzabi, Abdulhakem Y

2016-07-01

This article provides insight into the mechanism of femtosecond laser nanosurgical attachment of cells. We have demonstrated that during the attachment of two retinoblastoma cells using sub-10 femtosecond laser pulses, with 800 nm central wavelength, the phospholipid molecules of both cells hemifuse and form one shared phospholipid bilayer, at the attachment location. In order to verify the hypothesis that hemifusion takes place, transmission electron microscope images of the cell membranes of retinoblastoma cells were taken. It is shown that at the attachment interface, the two cell membranes coalesce and form one single membrane shared by both cells. Thus, further evidence is provided to support the hypothesis that laser-induced ionization process led to an ultrafast reversible destabilization of the phospholipid layer of the cellular membrane, which resulted in cross-linking of the phospholipid molecules in each membrane. This process of hemifusion occurs throughout the entire penetration depth of the femtosecond laser pulse train. Thus, the attachment between the cells takes place across a large surface area, which affirms our findings of strong physical attachment between the cells. The femtosecond laser pulse hemifusion technique can potentially provide a platform for precise molecular manipulation of cellular membranes. Manipulation of the cellular membrane is an important procedure that could aid in studying diseases such as cancer; where the expression level of plasma proteins on the cell membrane is altered.

Continuous inertial microparticle and blood cell separation in straight channels with local microstructures.

PubMed

Wu, Zhenlong; Chen, Yu; Wang, Moran; Chung, Aram J

2016-02-07

Fluid inertia which has conventionally been neglected in microfluidics has been gaining much attention for particle and cell manipulation because inertia-based methods inherently provide simple, passive, precise and high-throughput characteristics. Particularly, the inertial approach has been applied to blood separation for various biomedical research studies mainly using spiral microchannels. For higher throughput, parallelization is essential; however, it is difficult to realize using spiral channels because of their large two dimensional layouts. In this work, we present a novel inertial platform for continuous sheathless particle and blood cell separation in straight microchannels containing microstructures. Microstructures within straight channels exert secondary flows to manipulate particle positions similar to Dean flow in curved channels but with higher controllability. Through a balance between inertial lift force and microstructure-induced secondary flow, we deterministically position microspheres and cells based on their sizes to be separated downstream. Using our inertial platform, we successfully sorted microparticles and fractionized blood cells with high separation efficiencies, high purities and high throughputs. The inertial separation platform developed here can be operated to process diluted blood with a throughput of 10.8 mL min(-1)via radially arrayed single channels with one inlet and two rings of outlets.

Cells and Stripes: A novel quantitative photo-manipulation technique

PubMed Central

Mistrik, Martin; Vesela, Eva; Furst, Tomas; Hanzlikova, Hana; Frydrych, Ivo; Gursky, Jan; Majera, Dusana; Bartek, Jiri

2016-01-01

Laser micro-irradiation is a technology widely used in the DNA damage response, checkpoint signaling, chromatin remodeling and related research fields, to assess chromatin modifications and recruitment of diverse DNA damage sensors, mediators and repair proteins to sites of DNA lesions. While this approach has aided numerous discoveries related to cell biology, maintenance of genome integrity, aging and cancer, it has so far been limited by a tedious manual definition of laser-irradiated subcellular regions, with the ensuing restriction to only a small number of cells treated and analyzed in a single experiment. Here, we present an improved and versatile alternative to the micro-irradiation approach: Quantitative analysis of photo-manipulated samples using innovative settings of standard laser-scanning microscopes. Up to 200 cells are simultaneously exposed to a laser beam in a defined pattern of collinear rays. The induced striation pattern is then automatically evaluated by a simple algorithm, which provides a quantitative assessment of various laser-induced phenotypes in live or fixed cells. Overall, this new approach represents a more robust alternative to existing techniques, and provides a versatile tool for a wide range of applications in biomedicine. PMID:26777522

Thermometry in dielectrophoresis chips for contact-free cell handling

NASA Astrophysics Data System (ADS)

Jaeger, M. S.; Mueller, T.; Schnelle, T.

2007-01-01

Cell biology applications, protocols in immunology and stem cell research, require that individual cells are handled under strict control of their contacts to other cells or synthetic surfaces. Dielectrophoresis (DEP) in microfluidic chips is an established technique to investigate, group, wash, cultivate and sort cells contact-free under physiological conditions: microelectrode octode cages, versatile dielectrophoretic elements energized with radio frequency electric fields, stably trap single cells or cellular aggregates. For medical applications and cell cultivation, possible side effects of the dielectrophoretic manipulation, such as membrane polarization and Joule heating, have to be quantified. Therefore, we characterized the electric field-induced warming in dielectrophoretic cages using ohmic resistance measurements, fluorometry, liquid crystal beads, infra-red thermography and bubble size thermometry. We compare the results of these techniques with respect to the influences of voltage, electric conductivity of buffer, frequency, cage size and electrode surface. We conclude that in the culture medium thermal effects may be neglected if low voltages and an electric field-reducing phase pattern are used. Our experimental results provide explicit values for estimating the thermal effect on dielectrophoretically caged cells and show that Joule heating is best minimized by optimizing the cage geometry and reducing the buffer conductivity. The results may additionally serve to evaluate and improve theoretical predictions on field-induced effects. Based on present-day chip processing possibilities, DEP is well suited for the manipulation of cells.

Volumetric Stress-Strain Analysis of Optohydrodynamically Suspended Biological Cells

PubMed Central

Liang, Yu; Saha, Asit K.

2011-01-01

Ongoing investigations are exploring the biomechanical properties of isolated and suspended biological cells in pursuit of understanding single-cell mechanobiology. An optical tweezer with minimal applied laser power has positioned biologic cells at the geometric center of a microfluidic cross-junction, creating a novel optohydrodynamic trap. The resulting fluid flow environment facilitates unique multiaxial loading of single cells with site-specific normal and shear stresses resulting in a physical albeit extensional state. A recent two-dimensional analysis has explored the cytoskeletal strain response due to these fluid-induced stresses [Wilson and Kohles, 2010, “Two-Dimensional Modeling of Nanomechanical Stresses-Strains in Healthy and Diseased Single-Cells During Microfluidic Manipulation,” J Nanotechnol Eng Med, 1(2), p. 021005]. Results described a microfluidic environment having controlled nanometer and piconewton resolution. In this present study, computational fluid dynamics combined with multiphysics modeling has further characterized the applied fluid stress environment and the solid cellular strain response in three dimensions to accompany experimental cell stimulation. A volumetric stress-strain analysis was applied to representative living cell biomechanical data. The presented normal and shear stress surface maps will guide future microfluidic experiments as well as provide a framework for characterizing cytoskeletal structure influencing the stress to strain response. PMID:21186894

Microfluidic Blood Cell Preparation: Now and Beyond

PubMed Central

Yu, Zeta Tak For; Yong, Koh Meng Aw; Fu, Jianping

2014-01-01

Blood plays an important role in homeostatic regulation with each of its cellular components having important therapeutic and diagnostic uses. Therefore, separation and sorting of blood cells has been of a great interest to clinicians and researchers. However, while conventional methods of processing blood have been successful in generating relatively pure fractions, they are time consuming, labor intensive, and are not optimal for processing small volume blood samples. In recent years, microfluidics has garnered great interest from clinicians and researchers as a powerful technology for separating blood into different cell fractions. As microfluidics involves fluid manipulation at the microscale level, it has the potential for achieving high-resolution separation and sorting of blood cells down to a single-cell level, with an added benefit of integrating physical and biological methods for blood cell separation and analysis on the same single chip platform. This paper will first review the conventional methods of processing and sorting blood cells, followed by a discussion on how microfluidics is emerging as an efficient tool to rapidly change the field of blood cell sorting for blood-based therapeutic and diagnostic applications. PMID:24515899

MicroBioRobots for single cell manipulation

NASA Astrophysics Data System (ADS)

Sakar, Mahmut Selman

One of the great challenges in nano and micro scale science and engineering is the independent manipulation of biological cells and small man-made objects with active sensing. For such biomedical applications as single cell manipulation, telemetry, and localized targeted delivery of chemicals, it is important to fabricate microstructures that can be powered and controlled without a tether in fluidic environments. These microstructures can be used to develop microrobots that have the potential to make existing therapeutic and diagnostic procedures less invasive. Actuation can be realized using various different organic and inorganic methods. Previous studies explored different forms of actuation and control with microorganisms. Bacteria, in particular, offer several advantages as controllable microactuators: they draw chemical energy directly from their environment, they are genetically modifiable, and they are scalable and configurable in the sense that any number of bacteria can be selectively patterned. Additionally, the study of bacteria inspires inorganic schemes of actuation and control. For these reasons, we chose to employ bacteria while controlling their motility using optical and electrical stimuli. In the first part of the thesis, we demonstrate a biointegrated approach by introducing MicroBioRobots (MBRs). MBRs are negative photosensitive epoxy (SU8) microfabricated structures with typical feature sizes ranging from 1-100 mum coated with a monolayer of the swarming Serratia marcescens . The adherent bacterial cells naturally coordinate to propel the microstructures in fluidic environments which we call Self-Actuation. First, we demonstrate the control of MBRs using self-actuation, DC electric fields and ultra-violet radiation and develop an experimentally-validated mathematical model for the MBRs. This model allows us to to steer the MBR to any position and orientation in a planar micro channel using visual feedback and an inverted microscope. Examples of sub-micron scale transport and assembly as well as computer-based closed-loop control of MBRs are presented. We demonstrate experimentally that vision-based feedback control allows a four-electrode experimental device to steer MBRs along arbitrary paths with micrometer precision. At each time instant, the system identifies the current location of the robot, a control algorithm determines the power supply voltages that will move the charged robot from its current location toward its next desired position, and the necessary electric field is then created. Second, we develop biosensors for the MBRs. Microscopic devices with sensing capabilities could significantly improve single cell analysis, especially in high-resolution detection of patterns of chemicals released from cells in vitro. Two different types of sensing mechanisms are employed. The first method is based on harnessing bacterial power, and in the second method we use genetically engineered bacteria. The small size of the devices gives them access to individual cells, and their large numbers permit simultaneous monitoring of many cells. In the second part, we describe the construction and operation of truly micron-sized, biocompatible ferromagnetic micro transporters driven by external magnetic fields capable of exerting forces at the pico Newton scale. We develop micro transporters using a simple, single step micro fabrication technique that allows us to produce large numbers in the same step. We also fabricate microgels to deliver drugs. We demonstrate that the micro transporters can be navigated to separate single cells with micron-size precision and localize microgels without disturbing the local environment.

Cell refractive index for cell biology and disease diagnosis: past, present and future.

PubMed

Liu, P Y; Chin, L K; Ser, W; Chen, H F; Hsieh, C-M; Lee, C-H; Sung, K-B; Ayi, T C; Yap, P H; Liedberg, B; Wang, K; Bourouina, T; Leprince-Wang, Y

2016-02-21

Cell refractive index is a key biophysical parameter, which has been extensively studied. It is correlated with other cell biophysical properties including mechanical, electrical and optical properties, and not only represents the intracellular mass and concentration of a cell, but also provides important insight for various biological models. Measurement techniques developed earlier only measure the effective refractive index of a cell or a cell suspension, providing only limited information on cell refractive index and hence hindering its in-depth analysis and correlation. Recently, the emergence of microfluidic, photonic and imaging technologies has enabled the manipulation of a single cell and the 3D refractive index of a single cell down to sub-micron resolution, providing powerful tools to study cells based on refractive index. In this review, we provide an overview of cell refractive index models and measurement techniques including microfluidic chip-based techniques for the last 50 years, present the applications and significance of cell refractive index in cell biology, hematology, and pathology, and discuss future research trends in the field, including 3D imaging methods, integration with microfluidics and potential applications in new and breakthrough research areas.

Design of covalently functionalized carbon nanotubes filled with metal oxide nanoparticles for imaging, therapy, and magnetic manipulation.

PubMed

Liu, Xiaojie; Marangon, Iris; Melinte, Georgian; Wilhelm, Claire; Ménard-Moyon, Cécilia; Pichon, Benoit P; Ersen, Ovidiu; Aubertin, Kelly; Baaziz, Walid; Pham-Huu, Cuong; Bégin-Colin, Sylvie; Bianco, Alberto; Gazeau, Florence; Bégin, Dominique

2014-11-25

Nanocomposites combining multiple functionalities in one single nano-object hold great promise for biomedical applications. In this work, carbon nanotubes (CNTs) were filled with ferrite nanoparticles (NPs) to develop the magnetic manipulation of the nanotubes and their theranostic applications. The challenges were both the filling of CNTs with a high amount of magnetic NPs and their functionalization to form biocompatible water suspensions. We propose here a filling process using CNTs as nanoreactors for high-yield in situ growth of ferrite NPs into the inner carbon cavity. At first, NPs were formed inside the nanotubes by thermal decomposition of an iron stearate precursor. A second filling step was then performed with iron or cobalt stearate precursors to enhance the encapsulation yield and block the formed NPs inside the tubes. Water suspensions were then obtained by addition of amino groups via the covalent functionalization of the external surface of the nanotubes. Microstructural and magnetic characterizations confirmed the confinement of NPs into the anisotropic structure of CNTs making them suitable for magnetic manipulations and MRI detection. Interactions of highly water-dispersible CNTs with tumor cells could be modulated by magnetic fields without toxicity, allowing control of their orientation within the cell and inducing submicron magnetic stirring. The magnetic properties were also used to quantify CNTs cellular uptake by measuring the cell magnetophoretic mobility. Finally, the photothermal ablation of tumor cells could be enhanced by magnetic stimulus, harnessing the hybrid properties of NP loaded-CNTs.

Effect of Cell Sheet Manipulation Techniques on the Expression of Collagen Type II and Stress Fiber Formation in Human Chondrocyte Sheets.

PubMed

Wongin, Sopita; Waikakul, Saranatra; Chotiyarnwong, Pojchong; Siriwatwechakul, Wanwipa; Viravaidya-Pasuwat, Kwanchanok

2018-03-01

Cell sheet technology is applied to human articular chondrocytes to construct a tissue-like structure as an alternative treatment for cartilage defect. The effect of a gelatin manipulator, as a cell sheet transfer system, on the quality of the chondrocyte sheets was investigated. The changes of important chondrogenic markers and stress fibers, resulting from the cell sheet manipulation, were also studied. The chondrocyte cell sheets were constructed with patient-derived chondrocytes using a temperature-responsive polymer and a gelatin manipulator as a transfer carrier. The properties of the cell sheets, including sizes, expression levels of collagen type II and I, and the localization of the stress fibers, were assessed and compared with those of the cell sheets harvested without the gelatin manipulator. Using the gelatin manipulator, the original size of the chondrocyte cell sheets was retained with abundant stress fibers, but with a decrease in the expression of collagen type II. Without the gelatin manipulator, although the cell shrinkage occurred, the cell sheet with suppressed stress fiber formation showed significantly higher levels of collagen type II. These results support our observations that stress fiber formation in chondrocyte cell sheets affected the production of chondrogenic markers. These densely packed tissue-like structures possessed a good chondrogenic activity, indicating their potential for use in autologous chondrocyte implantation to treat cartilage defects.

Applications and Limitations of Micropropagation for the Production of Underwater Grasses

DTIC Science & Technology

2006-01-01

ERDC/TN SAV-06-1 January 2006 Applications and Limitations of Micropropagation for the Production of Underwater Grasses by Steve Ailstock and...Deborah Shafer DEFINITION: Micropropagation is a technique that manipulates small quantities of axenic plant mate- rial, ranging from single cells to...plant structures. Micropropagation , on the other hand, allows the year-round production of new plants at rates significantly higher than that

Magnetoelectricity in Multi-Scale Composites and Application in Nanorobotics for Live Cell Manipulation

NASA Astrophysics Data System (ADS)

Betal, Soutik

In this research biomedical and sensor applications of magnetoelectric effect have been broadly explored using magnetoelectric composites. Firstly NiFe2O4/Pb(Zr0.52Ti0.48)O 3/NiFe2O4 layered bulk composite have been studied to achieve high magnetoelectric coefficient for their applications in brain magnetic field detection at room temperature. Magnetic sensors like SQUID (superconducting quantum interference device) nowadays are able to detect pico-Tesla magnetic fields produced outside the brain by the neuronal currents which can be used for diagnostic application, but due to heavy liquid helium cooling and insulation requirements, the technique become quite inefficient in gaining high resolution measurement. At room temperature layered ME samples exhibit high magnetoelectric response in mV/cm.Oe range and hence can transform very low magnetic field into electric signal which can be measured even in femtovolts. Moreover temperature and a.c. frequency dependent studies were done to extensively characterize the layered ME sample for sensor application. Secondly core-shell magnetoelectric nanoparticles (CSMEN) have been fabricated, characterized and their interaction with biological cell in presence of a.c. and d.c. field have been thoroughly analyzed. A magnetically controlled elastically driven electroporation phenomenon, or Magneto-Elasto- Electroporation (MEEP), is discovered while studying interactions between core-shell magneto-electric nanoparticles (CSMEN) and biological cells in the presence of an AC magnetic field. In this research MEEP effect was observed via a series of in-vitro experiments using core (CoFe2O4)-shell (BaTiO3 ) structured magnetoelectric nanoparticles and human epithelial cells (HEP2). Cell electroporation phenomenon and its correlation with the magnetic field modulated CSMEN have been elaborately studied. Potential of CSMEN for application in targeted single cell electroporation have been confirmed by analysing crystallographic phases, multiferroic properties of the fabricated CSMEN , influences of DC and AC magnetic field on the CSMEN and cytotoxicity tests. We also report the mathematical formalism to quantitatively describe the phenomena. The reported findings provide the basis of the underlying MEEP mechanism and demonstrate the utility of CSMEN as electric pulse generating nano-probe in cell electroporation experiments for the potential application towards accurate and efficient targeted cell permeation as well as drug delivery. Thirdly, experiments of fabricated magnetoelectric nanocomposites with biological cells in controlled boundary condition under fluctuating and biased magnetic field excitation revealed the smart nanorobotics characteristics of the nanostructure to achieve remote controlled dynamically targeted live cell manipulation. A remotely controlled dynamic process of manipulating targeted biological live cells using fabricated core-shell magnetoelectric nanocomposites have been fabricated, which comprises of single crystalline ferromagnetic cores (CoFe2O4) coated with crystalline ferroelectric thin film shells (BaTiO3). These nanocomposites are demonstrated as a unique family of inorganic magnetoelectric nanorobots (MENRs), controlled remotely by applied a.c. or d.c. magnetic fields, to perform cell targeting, permeation, patterning and transport. MENRs performs these functions via localized electric periodic pulse generation, local electric-field sensing, or thrust generation and acts as a unique tool for remotely controlled dynamically targeted cellular manipulation. Under a.c. magnetic field excitation (50 Oe, 60 Hz), the MENR acts as a localized periodic electric pulse generator and can permeate a series of misaligned cells, while aligning/patterning them to an equipotential mono-array. Under a.c. magnetic field (40 Oe, 30 Hz) excitation, MENRs can be dynamically driven to a targeted cell, avoiding untargeted cells in the path, irrespective of cell density. D.C. magnetic field (-50 Oe) excitation causes the MENRs to act as thrust generator and exerts motion in a group of cells. Visualization of magnetoelectricity at nanoscale and its application in dynamically targeted live cell manipulation have been presented in this research.

Single-cell quantification of IL-2 response by effector and regulatory T cells reveals critical plasticity in immune response

PubMed Central

Feinerman, Ofer; Jentsch, Garrit; Tkach, Karen E; Coward, Jesse W; Hathorn, Matthew M; Sneddon, Michael W; Emonet, Thierry; Smith, Kendall A; Altan-Bonnet, Grégoire

2010-01-01

Understanding how the immune system decides between tolerance and activation by antigens requires addressing cytokine regulation as a highly dynamic process. We quantified the dynamics of interleukin-2 (IL-2) signaling in a population of T cells during an immune response by combining in silico modeling and single-cell measurements in vitro. We demonstrate that IL-2 receptor expression levels vary widely among T cells creating a large variability in the ability of the individual cells to consume, produce and participate in IL-2 signaling within the population. Our model reveals that at the population level, these heterogeneous cells are engaged in a tug-of-war for IL-2 between regulatory (Treg) and effector (Teff) T cells, whereby access to IL-2 can either increase the survival of Teff cells or the suppressive capacity of Treg cells. This tug-of-war is the mechanism enforcing, at the systems level, a core function of Treg cells, namely the specific suppression of survival signals for weakly activated Teff cells but not for strongly activated cells. Our integrated model yields quantitative, experimentally validated predictions for the manipulation of Treg suppression. PMID:21119631

Manipulation of mammalian cells using a single-fiber optical microbeam

PubMed Central

Mohanty, Samarendra K.; Mohanty, Khyati S.; Berns, Michael W.

2014-01-01

The short working distance of microscope objectives has severely restricted the application of optical micromanipulation techniques at larger depths. We show the first use of fiber-optic tweezers toward controlled guidance of neuronal growth cones and stretching of neurons. Further, by mode locking, the fiber-optic tweezers beam was converted to fiber-optic scissors, enabling dissection of neuronal processes and thus allowing study of the subsequent response of neurons to localized injury. At high average powers, lysis of a three-dimensionally trapped cell was accomplished. PMID:19021429

Variability in Cell Response of Cronobacter sakazakii after Mild-Heat Treatments and Its Impact on Food Safety

PubMed Central

Parra-Flores, Julio; Juneja, Vijay; Garcia de Fernando, Gonzalo; Aguirre, Juan

2016-01-01

Cronobacter spp. have been responsible for severe infections in infants associated with consumption of powdered infant formula and follow-up formulae. Despite several risk assessments described in published studies, few approaches have considered the tremendous variability in cell response that small micropopulations or single cells can have in infant formula during storage, preparation or post process/preparation before the feeding of infants. Stochastic approaches can better describe microbial single cell response than deterministic models as we prove in this study. A large variability of lag phase was observed in single cell and micropopulations of ≤50 cells. This variability increased as the heat shock increased and growth temperature decreased. Obviously, variability of growth of individual Cronobacter sakazakii cell is affected by inoculum size, growth temperature and the probability of cells able to grow at the conditions imposed by the experimental conditions should be taken into account, especially when errors in bottle-preparation practices, such as improper holding temperatures, or manipulation, may lead to growth of the pathogen to a critical cell level. The mean probability of illness from initial inoculum size of 1 cell was below 0.2 in all the cases and for inoculum size of 50 cells the mean probability of illness, in most of the cases, was above 0.7. PMID:27148223

Manipulating, Reacting, and Constructing Single Molecules with a Scanning Tunneling Microscope Tip

NASA Astrophysics Data System (ADS)

Hla, S.-W.

The fascinating advances in atom and molecule manipulation with the scanning tunneling microscope (STM) tip allow scientists to fabricate artificial atomic scale structures, to study local quantum phenomena, or to probe physical and chemical properties of single atoms and molecules on surfaces. Recent achievements in individual synthesis of single molecules with the STM tip further open up an entirely new opportunities in nanoscience and technology. The STM manipulation techniques usef ul in the molecular construction are reviewed and prospects for future opportunities of single molecule chemical engineering and their possible implications to nano-scale science and technology are discussed.

Immuno-Oncology-The Translational Runway for Gene Therapy: Gene Therapeutics to Address Multiple Immune Targets.

PubMed

Weß, Ludger; Schnieders, Frank

2017-12-01

Cancer therapy is once again experiencing a paradigm shift. This shift is based on extensive clinical experience demonstrating that cancer cannot be successfully fought by addressing only single targets or pathways. Even the combination of several neo-antigens in cancer vaccines is not sufficient for successful, lasting tumor eradication. The focus has therefore shifted to the immune system's role in cancer and the striking abilities of cancer cells to manipulate and/or deactivate the immune system. Researchers and pharma companies have started to target the processes and cells known to support immune surveillance and the elimination of tumor cells. Immune processes, however, require novel concepts beyond the traditional "single-target-single drug" paradigm and need parallel targeting of diverse cells and mechanisms. This review gives a perspective on the role of gene therapy technologies in the evolving immuno-oncology space and identifies gene therapy as a major driver in the development and regulation of effective cancer immunotherapy. Present challenges and breakthroughs ranging from chimeric antigen receptor T-cell therapy, gene-modified oncolytic viruses, combination cancer vaccines, to RNA therapeutics are spotlighted. Gene therapy is recognized as the most prominent technology enabling effective immuno-oncology strategies.

Single cell integration of animate form, motion and location in the superior temporal cortex of the macaque monkey.

PubMed

Jellema, Tjeerd; Maassen, Gerard; Perrett, David I

2004-07-01

This study investigated the cellular mechanisms in the anterior part of the superior temporal sulcus (STSa) that underlie the integration of different features of the same visually perceived animate object. Three visual features were systematically manipulated: form, motion and location. In 58% of a population of cells selectively responsive to the sight of a walking agent, the location of the agent significantly influenced the cell's response. The influence of position was often evident in intricate two- and three-way interactions with the factors form and/or motion. For only one of the 31 cells tested, the response could be explained by just a single factor. For all other cells at least two factors, and for half of the cells (52%) all three factors, played a significant role in controlling responses. Our findings support a reformulation of the Ungerleider and Mishkin model, which envisages a subdivision of the visual processing into a ventral 'what' and a dorsal 'where' stream. We demonstrated that at least part of the temporal cortex ('what' stream) makes ample use of visual spatial information. Our findings open up the prospect of a much more elaborate integration of visual properties of animate objects at the single cell level. Such integration may support the comprehension of animals and their actions.

Probing cellular mechanics with Acoustic Force Spectroscopy.

PubMed

Sorkin, Raya; Bergamaschi, Giulia; Kamsma, Douwe; Brand, Guy; Dekel, Elya; Ofir-Birin, Yifat; Rudik, Ariel; Gironella, Marta; Ritort, Felix; Regev-Rudzki, Neta; Roos, Wouter; Wuite, Gijs J L

2018-06-21

A large number of studies demonstrate that cell mechanics and pathology are intimately linked. In particular, Red Blood Cell (RBC) deformability is key to their function, and is dramatically altered in the time course of diseases such as anemia and malaria. Due to the physiological importance of cell mechanics, many methods for cell mechanical probing have been developed. While single cell methods provide very valuable information, they are often technically challenging and lack high data throughput needed to distinguish differences in heterogeneous populations, while fluid flow high throughput methods miss the accuracy to detect subtle differences. Here, we present a new method for multiplexed single-cell mechanical probing using Acoustic Force Spectroscopy (AFS). We demonstrate that mechanical differences induced by chemical treatments of known effect can be measured and quantified. Furthermore, we explore the effect of extracellular-vesicles (EVs) uptake on RBC mechanics and demonstrate that EVs uptake increases RBC deformability. Our findings demonstrate the ability of AFS to manipulate cells with high stability and precision, and pave the way to further new insights into cellular mechanics and mechanobiology in health and disease, as well as potential biomedical applications.

The glass micropipette electrode: A history of its inventors and users to 1950

PubMed Central

2017-01-01

Soon after the glass micropipette was invented as a micro-tool for manipulation of single bacteria and the microinjection and microsurgery of living cells, it was seen to hold promise as a microelectrode to stimulate individual cells electrically and to study electrical potentials in them. Initial successes and accurate mechanistic explanations of the results were achieved in giant plant cells in the 1920s. Long known surface electrical activity in nerves and muscles was only resolved at a similar cellular level in the 1930s and 1940s after the discovery of giant nerve fibers and the development of finer tipped microelectrodes for normal-sized cells. PMID:28298356

Bilateral and multiple cavitation sounds during upper cervical thrust manipulation

PubMed Central

2013-01-01

Background The popping produced during high-velocity, low-amplitude (HVLA) thrust manipulation is a common sound; however to our knowledge, no study has previously investigated the location of cavitation sounds during manipulation of the upper cervical spine. The primary purpose was to determine which side of the spine cavitates during C1-2 rotatory HVLA thrust manipulation. Secondary aims were to calculate the average number of pops, the duration of upper cervical thrust manipulation, and the duration of a single cavitation. Methods Nineteen asymptomatic participants received two upper cervical thrust manipulations targeting the right and left C1-2 articulation, respectively. Skin mounted microphones were secured bilaterally over the transverse process of C1, and sound wave signals were recorded. Identification of the side, duration, and number of popping sounds were determined by simultaneous analysis of spectrograms with audio feedback using custom software developed in Matlab. Results Bilateral popping sounds were detected in 34 (91.9%) of 37 manipulations while unilateral popping sounds were detected in just 3 (8.1%) manipulations; that is, cavitation was significantly (P < 0.001) more likely to occur bilaterally than unilaterally. Of the 132 total cavitations, 72 occurred ipsilateral and 60 occurred contralateral to the targeted C1-2 articulation. In other words, cavitation was no more likely to occur on the ipsilateral than the contralateral side (P = 0.294). The mean number of pops per C1-2 rotatory HVLA thrust manipulation was 3.57 (95% CI: 3.19, 3.94) and the mean number of pops per subject following both right and left C1-2 thrust manipulations was 6.95 (95% CI: 6.11, 7.79). The mean duration of a single audible pop was 5.66 ms (95% CI: 5.36, 5.96) and the mean duration of a single manipulation was 96.95 ms (95% CI: 57.20, 136.71). Conclusions Cavitation was significantly more likely to occur bilaterally than unilaterally during upper cervical HVLA thrust manipulation. Most subjects produced 3–4 pops during a single rotatory HVLA thrust manipulation targeting the right or left C1-2 articulation; therefore, practitioners of spinal manipulative therapy should expect multiple popping sounds when performing upper cervical thrust manipulation to the atlanto-axial joint. Furthermore, the traditional manual therapy approach of targeting a single ipsilateral or contralateral facet joint in the upper cervical spine may not be realistic. PMID:23320608

Combined acoustic and optical trapping

PubMed Central

Thalhammer, G.; Steiger, R.; Meinschad, M.; Hill, M.; Bernet, S.; Ritsch-Marte, M.

2011-01-01

Combining several methods for contact free micro-manipulation of small particles such as cells or micro-organisms provides the advantages of each method in a single setup. Optical tweezers, which employ focused laser beams, offer very precise and selective handling of single particles. On the other hand, acoustic trapping with wavelengths of about 1 mm allows the simultaneous trapping of many, comparatively large particles. With conventional approaches it is difficult to fully employ the strengths of each method due to the different experimental requirements. Here we present the combined optical and acoustic trapping of motile micro-organisms in a microfluidic environment, utilizing optical macro-tweezers, which offer a large field of view and working distance of several millimeters and therefore match the typical range of acoustic trapping. We characterize the acoustic trapping forces with the help of optically trapped particles and present several applications of the combined optical and acoustic trapping, such as manipulation of large (75 μm) particles and active particle sorting. PMID:22025990

Multimodal biophotonic workstation for live cell analysis.

PubMed

Esseling, Michael; Kemper, Björn; Antkowiak, Maciej; Stevenson, David J; Chaudet, Lionel; Neil, Mark A A; French, Paul W; von Bally, Gert; Dholakia, Kishan; Denz, Cornelia

2012-01-01

A reliable description and quantification of the complex physiology and reactions of living cells requires a multimodal analysis with various measurement techniques. We have investigated the integration of different techniques into a biophotonic workstation that can provide biological researchers with these capabilities. The combination of a micromanipulation tool with three different imaging principles is accomplished in a single inverted microscope which makes the results from all the techniques directly comparable. Chinese Hamster Ovary (CHO) cells were manipulated by optical tweezers while the feedback was directly analyzed by fluorescence lifetime imaging, digital holographic microscopy and dynamic phase-contrast microscopy. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[A skin cell segregating control system based on PC].

PubMed

Liu, Wen-zhong; Zhou, Ming; Zhang, Hong-bing

2005-11-01

A skin cell segregating control system based on PC (personal computer) is presented in this paper. Its front controller is a single-chip microcomputer which enables the manipulation for 6 patients simultaneously, and thus provides a great convenience for clinical treatments for vitiligo. With the use of serial port communication technology, it's possible to monitor and control the front controller in a PC terminal. And the application of computer image acquisition technology realizes the synchronous acquisition of pathologic shin cell images pre/after the operation and a case history. Clinical tests prove its conformity with national standards and the pre-set technological requirements.

Nano/micro-scale magnetophoretic devices for biomedical applications

NASA Astrophysics Data System (ADS)

Lim, Byeonghwa; Vavassori, Paolo; Sooryakumar, R.; Kim, CheolGi

2017-01-01

In recent years there have been tremendous advances in the versatility of magnetic shuttle technology using nano/micro-scale magnets for digital magnetophoresis. While the technology has been used for a wide variety of single-cell manipulation tasks such as selection, capture, transport, encapsulation, transfection, or lysing of magnetically labeled and unlabeled cells, it has also expanded to include parallel actuation and study of multiple bio-entities. The use of nano/micro-patterned magnetic structures that enable remote control of the applied forces has greatly facilitated integration of the technology with microfluidics, thereby fostering applications in the biomedical arena. The basic design and fabrication of various scaled magnets for remote manipulation of individual and multiple beads/cells, and their associated energies and forces that underlie the broad functionalities of this approach, are presented. One of the most useful features enabled by such advanced integrated engineering is the capacity to remotely tune the magnetic field gradient and energy landscape, permitting such multipurpose shuttles to be implemented within lab-on-chip platforms for a wide range of applications at the intersection of cellular biology and biotechnology.

Rational Manipulation of the Standard Laparoscopic Instruments for Single-Incision Laparoscopic Right Colectomy

PubMed Central

Watanabe, Makoto; Murakami, Masahiko; Kato, Takashi; Onaka, Toru; Aoki, Takeshi

2013-01-01

This report clarifies the rational manipulation of standard laparoscopic instruments for single-incision laparoscopic right colectomy (SILRC) using the SILS Port. We classified the manipulations required into 4 techniques. Vertical manipulation was required for medial-to-lateral retroperitoneal dissection. Frontal manipulation was needed for extension and establishment of a retroperitoneal plane. External crossing manipulation was used for dissection or ligation of the ileocolic or right colic vessels. Internal crossing manipulation was required for mobilization from the cecum to ascending colon. We performed SILRC for a series of 30 consecutive patients. One additional port was needed in 5 of the patients (16.7%) because of severe adhesion between the ileum and abdominal wall. No intraoperative complications were encountered. Four rational manipulations of the standard laparoscopic instruments are required for SILRC using the SILS Port. However, more experience and comparative trials are needed to determine the exact role of SILRC. PMID:23971771

Control of a flexible bracing manipulator: Integration of current research work to realize the bracing manipulator

NASA Technical Reports Server (NTRS)

Kwon, Dong-Soo

1991-01-01

All research results about flexible manipulator control were integrated to show a control scenario of a bracing manipulator. First, dynamic analysis of a flexible manipulator was done for modeling. Second, from the dynamic model, the inverse dynamic equation was derived, and the time domain inverse dynamic method was proposed for the calculation of the feedforward torque and the desired flexible coordinate trajectories. Third, a tracking controller was designed by combining the inverse dynamic feedforward control with the joint feedback control. The control scheme was applied to the tip position control of a single link flexible manipulator for zero and non-zero initial condition cases. Finally, the contact control scheme was added to the position tracking control. A control scenario of a bracing manipulator is provided and evaluated through simulation and experiment on a single link flexible manipulator.

Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping

PubMed Central

Normanno, Davide; Vanzi, Francesco; Pavone, Francesco Saverio

2008-01-01

Gene expression regulation is a fundamental biological process which deploys specific sets of genomic information depending on physiological or environmental conditions. Several transcription factors (including lac repressor, LacI) are present in the cell at very low copy number and increase their local concentration by binding to multiple sites on DNA and looping the intervening sequence. In this work, we employ single-molecule manipulation to experimentally address the role of DNA supercoiling in the dynamics and stability of LacI-mediated DNA looping. We performed measurements over a range of degrees of supercoiling between −0.026 and +0.026, in the absence of axial stretching forces. A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed. Our experiments also provide evidence for multiple structural conformations of the LacI–DNA complex, depending on torsional constraints. The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon. In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes. PMID:18310101

Watching cellular machinery in action, one molecule at a time.

PubMed

Monachino, Enrico; Spenkelink, Lisanne M; van Oijen, Antoine M

2017-01-02

Single-molecule manipulation and imaging techniques have become important elements of the biologist's toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components. © 2017 Monachino et al.

Acoustic devices for particle and cell manipulation and sensing.

PubMed

Qiu, Yongqiang; Wang, Han; Demore, Christine E M; Hughes, David A; Glynne-Jones, Peter; Gebhardt, Sylvia; Bolhovitins, Aleksandrs; Poltarjonoks, Romans; Weijer, Kees; Schönecker, Andreas; Hill, Martyn; Cochran, Sandy

2014-08-13

An emerging demand for the precise manipulation of cells and particles for applications in cell biology and analytical chemistry has driven rapid development of ultrasonic manipulation technology. Compared to the other manipulation technologies, such as magnetic tweezing, dielectrophoresis and optical tweezing, ultrasonic manipulation has shown potential in a variety of applications, with its advantages of versatile, inexpensive and easy integration into microfluidic systems, maintenance of cell viability, and generation of sufficient forces to handle particles, cells and their agglomerates. This article briefly reviews current practice and reports our development of various ultrasonic standing wave manipulation devices, including simple devices integrated with high frequency (>20 MHz) ultrasonic transducers for the investigation of biological cells and complex ultrasonic transducer array systems to explore the feasibility of electronically controlled 2-D and 3-D manipulation. Piezoelectric and passive materials, fabrication techniques, characterization methods and possible applications are discussed. The behavior and performance of the devices have been investigated and predicted with computer simulations, and verified experimentally. Issues met during development are highlighted and discussed. To assist long term practical adoption, approaches to low-cost, wafer level batch-production and commercialization potential are also addressed.

Acoustic Devices for Particle and Cell Manipulation and Sensing

PubMed Central

Qiu, Yongqiang; Wang, Han; Demore, Christine E. M.; Hughes, David A.; Glynne-Jones, Peter; Gebhardt, Sylvia; Bolhovitins, Aleksandrs; Poltarjonoks, Romans; Weijer, Kees; Schönecker, Andreas; Hill, Martyn; Cochran, Sandy

2014-01-01

An emerging demand for the precise manipulation of cells and particles for applications in cell biology and analytical chemistry has driven rapid development of ultrasonic manipulation technology. Compared to the other manipulation technologies, such as magnetic tweezing, dielectrophoresis and optical tweezing, ultrasonic manipulation has shown potential in a variety of applications, with its advantages of versatile, inexpensive and easy integration into microfluidic systems, maintenance of cell viability, and generation of sufficient forces to handle particles, cells and their agglomerates. This article briefly reviews current practice and reports our development of various ultrasonic standing wave manipulation devices, including simple devices integrated with high frequency (>20 MHz) ultrasonic transducers for the investigation of biological cells and complex ultrasonic transducer array systems to explore the feasibility of electronically controlled 2-D and 3-D manipulation. Piezoelectric and passive materials, fabrication techniques, characterization methods and possible applications are discussed. The behavior and performance of the devices have been investigated and predicted with computer simulations, and verified experimentally. Issues met during development are highlighted and discussed. To assist long term practical adoption, approaches to low-cost, wafer level batch-production and commercialization potential are also addressed. PMID:25123465

Mechanical Coupling of Smooth Muscle Cells Using Microengineered Substrates and Local Stimulation

NASA Astrophysics Data System (ADS)

Copeland, Craig; Hunter, David; Tung, Leslie; Chen, Christopher; Reich, Daniel

2013-03-01

Mechanical stresses directly affect many cellular processes, including signal transduction, growth, differentiation, and survival. Cells can themselves generate such stresses by activating myosin to contract the actin cytoskeleton, which in turn can regulate both cell-substrate and cell-cell interactions. We are studying mechanical forces at cell-cell and cell-substrate interactions using arrays of selectively patterned flexible PDMS microposts combined with the ability to apply local chemical stimulation. Micropipette ``spritzing'', a laminar flow technique, uses glass micropipettes mounted on a microscope stage to deliver drugs to controlled regions within a cellular construct while cell traction forces are recorded via the micropost array. The pipettes are controlled by micromanipulators allowing for rapid and precise movement across the array and the ability to treat multiple constructs within a sample. This technique allows for observing the propagation of a chemically induced mechanical stimulus through cell-cell and cell-substrate interactions. We have used this system to administer the acto-myosin inhibitors Blebbistatin and Y-27632 to single cells and observed the subsequent decrease in cell traction forces. Experiments using trypsin-EDTA have shown this system to be capable of single cell manipulation through removal of one cell within a pair configuration while leaving the other cell unaffected. This project is supported in part by NIH grant HL090747

Bottom-up on-crystal in-chip formation of a conducting salt and a view of its restructuring: from organic insulator to conducting "switch" through microfluidic manipulation.

PubMed

Puigmartí-Luis, Josep; Paradinas, Markos; Bailo, Elena; Rodriguez-Trujillo, Romen; Pfattner, Raphael; Ocal, Carmen; Amabilino, David B

2015-06-01

The chemical modification of an immobilized single crystal in a fluid cell is reported, whereby a material with switching functions is generated in situ by generating a chemical reagent in the flow. Crystals of the insulating organic crystal of TCNQ (tetracyanoquinodimethane) were grown in a microfluidic channel and were trapped using a pneumatic valve, a nascent technique for materials manipulation. They were subsequently reduced using solution-deposited silver to provide a conducting material in situ by a heterogeneous reaction. Removal of the new material from the chip proved it to be the silver salt of reduced TCNQ. Uniquely, conducting atomic force microscope (CAFM) studies show three regions in the solid. The localized original neutral organic material crystal is shown to be an insulator but to produce areas with Ohmic conducting characteristics after reduction. This inhomogeneous doping provides an opportunity for probing electrical materials properties side by side. Measurements with the CAFM witness this conducting material where the TCNQ is fully transformed to the silver salt. Additionally, an intermediate phase is observed that exhibits bipolar resistive switching typical of programmable resistive memories. Raman microscopy proves the conversion of the material in specific regions and clearly defines the intermediate phase region that could be responsible for the switching effect in related materials. This kind of "on crystal chemistry" exploiting immobilization and masking by a pneumatic clamp in a microfluidic channel shows how material can be selectively converted to give different functionalities in the same material piece, even though it is not a single crystal to single crystal conversion, and beckons exploitation for the preparation of systems relevant for molecular electronics as well as other areas where chemical manipulation of single crystals could be beneficial.

Practical cell labeling with magnetite cationic liposomes for cell manipulation.

PubMed

Ito, Hiroshi; Nonogaki, Yurika; Kato, Ryuji; Honda, Hiroyuki

2010-07-01

Personalization of the cell culture process for cell therapy is an ideal strategy to obtain maximum treatment effects. In a previous report, we proposed a strategy using a magnetic manipulation device that combined a palm-top size device and a cell-labeling method using magnetite cationic liposomes (MCLs) to enable feasible personalized cell processing. In the present study, we focused on optimizing the MCL-labeling technique with respect to cell manipulation in small devices. From detailed analysis with different cell types, 4 pg/cell of MCL-label was found to be obtained immediately after mixing with MCLs, which was sufficient for magnetic cell manipulation. The amount of label increased within 24 h depending on cell type, although in all cases it decreased along with cell doubling, indicating that the labeling potential of MCLs was limited. The role of free MCLs not involved in labeling was also investigated; MCLs' role was found to be a supportive one that maximized the manipulation performance up to 100%. We also determined optimum conditions to manipulate adherent cells by MCL labeling using the MCL dispersed in trypsin solution. Considering labeling feasibility and practical performance with 10(3)-10(5) cells for personalized cell processing, we determined that 10 microg/ml of label without incubation time (0 h incubation) was the universal MCL-labeling condition. We propose the optimum specifications for a device to be combined with this method. 2010. Published by Elsevier B.V.

In situ flat embedding of monolayers and cell relocation in the acrylic resin LR white for comparative light and electron microscopy studies.

PubMed

Steiner, M; Schöfer, C; Mosgoeller, W

1994-12-01

A simple and reliable method has been developed for the in situ LR White embedding of cell monolayers grown on glass cover-slips. Combined with cytochemical or immunological procedures, this technique allows light and/or electron microscopy investigations of a large number of cells in the same horizontal plane within a relatively short period of time. It can be applied to cells grown on microgrid finder cover-slips which allows a distinct site of even an individual cell of a monolayer to be studied at first at the light microscope level and subsequently at the electron microscope level. Hence, it is also suitable for controlling manipulation of single cells, followed by their serial sectioning after relocation in the electron microscope.

Paper-based device for separation and cultivation of single microalga.

PubMed

Chen, Chih-Chung; Liu, Yi-Ju; Yao, Da-Jeng

2015-12-01

Single-cell separation is among the most useful techniques in biochemical research, diagnosis and various industrial applications. Microalgae species have great economic importance as industrial raw materials. Microalgae species collected from environment are typically a mixed and heterogeneous population of species that must be isolated and purified for examination and further application. Conventional methods, such as serial dilution and a streaking-plate method, are intensive of labor and inefficient. We developed a paper-based device for separation and cultivation of single microalga. The fabrication was simply conducted with a common laser printer and required only a few minutes without lithographic instruments and clean-room. The driving force of the paper device was simple capillarity without a complicated pump connection that is part of most devices for microfluidics. The open-structure design of the paper device makes it operable with a common laboratory micropipette for sample transfer and manipulation with a naked eye or adaptable to a robotic system with functionality of high-throughput retrieval and analysis. The efficiency of isolating a single cell from mixed microalgae species is seven times as great as with a conventional method involving serial dilution. The paper device can serve also as an incubator for microalgae growth on simply rinsing the paper with a growth medium. Many applications such as highly expressed cell selection and various single-cell analysis would be applicable. Copyright © 2015 Elsevier B.V. All rights reserved.

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

PubMed

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.

Avian germplasm preservation: embryonic stem cells or primordial germ cells?

PubMed

Petitte, J N

2006-02-01

Presently, avian genetic resources are best maintained as living collections of birds. Unfortunately, these stocks have been under constant pressure to be destroyed because of the decline in the number of Poultry Science Departments and pressures to cut costs at land grant institutions. Cryopreservation of semen is often suggested as a means to bank avian germplasm. However, this is only applicable for single-gene traits and does not allow for full reconstitution of the genetics of the original line. Over the last 15 yr, advances in the manipulation of the early chick embryo, manipulation of primordial germ cells (PGC), and the culture of embryonic stem cells (ESC) suggests that cryopreservation of blastodermal cells, ESC, or PGC might offer a means to preserve the entire genome of highly selected, specialized stocks of poultry. Freezing each of these cell types is possible with varying degrees of efficiency. Similarly, the effectiveness of generating germ line chimeras using blastodermal cells, ESC, or PGC also varies greatly. Other factors that must be considered include the choice of the recipient lines to develop the germ line chimeras and the number of individuals needed to reconstitute the line. Finally, the low efficiency rate of reconstitution and the high cost associated with current technologies makes these approaches prohibitive. Significant challenges remain to be overcome before the entire genome of poultry stocks can be routinely cryoperserved and reconstituted.

A polymeric micro total analysis system for single-cell analysis

NASA Astrophysics Data System (ADS)

Lai, Hsuan-Hong

The advancement of microengineering has enabled the manipulation and analysis of single cells, which is critical in understanding the molecular mechanisms underlying the basic physiological functions from the point of view of modern biologists. Unfortunately, analysis of single cells remains challenging from a technical perspective, mainly because of the miniature nature of the cell and the high throughput requirements of the analysis. Lab-on-a-chip (LOC) emerges as a research field that shows great promise in this perspective. We have demonstrated a micro total analysis system (mu-TAS) combining chip-based electrophoretic separation, fluorescence detection, and a pulsed Nd:YAG laser cell lysis system, in a Poly(dimethylsiloxane) (PDMS) microfluidic analytical platform for the implementation of single-cell analysis. To accomplish the task, a polymeric microfluidic device was fabricated and UV graft polymerization surface modification techniques were used. To optimize the conditions for the surface treatment techniques, the modified surfaces of PDMS were characterized using AIR-IR spectrum and sessile water drop contact angle measurements, and in-channel surfaces were characterized by their electroosmotic flow mobility. Accurate single-cell analysis relies on rapid cell lysis and therefore an optical measure of fast cell lysis was implemented and optimized in a microscopic station. The influences of pulse energy and the location of the laser beam with respect to the cell in the microchannel were explored. The observation from the cell disruption experiments suggested that the cell lysis was enabled mainly via a thermo-mechanical instead of a plasma-mediated mechanism. Finally, after chip-based electrophoresis and a laser-induced fluorescence (LIF) detection system were incorporated with the laser lysis system in a microfluidic analytical station, a feasibility demonstration of single-cell analysis was implemented. The analytical platform exhibited the capability of fluidic transportation, optical lysis of single cells, separation, and analysis of the lysates by electrophoresis and LIF detection. In comparison with the control experiment, the migration times of the fluorescent signals for the cytosolic fluorophores were in good agreement with those for the standard fluorophores, which confirmed the feasibility of the analytical processes.

Soft, flexible micromanipulators comprising polypyrrole trilayer microactuators

NASA Astrophysics Data System (ADS)

Khaldi, Alexandre; Maziz, Ali; Alici, Gursel; Spinks, Geoffrey M.; Jager, Edwin W. H.

2015-04-01

Within the areas of cell biology, biomedicine and minimal invasive surgery, there is a need for soft, flexible and dextrous biocompatible manipulators for handling biological objects, such as single cells and tissues. Present day technologies are based on simple suction using micropipettes for grasping objects. The micropipettes lack the possibility of accurate force control, nor are they soft and compliant and may thus cause damage to the cells or tissue. Other micromanipulators use conventional electric motors however the further miniaturization of electrical motors and their associated gear boxes and/or push/pull wires has reached its limits. Therefore there is an urgent need for new technologies for micromanipulation of soft biological matter. We are developing soft, flexible micromanipulators such as micro- tweezers for the handling and manipulation of biological species including cells and surgical tools for minimal invasive surgery. Our aim is to produce tools with minimal dimensions of 100 μm to 1 mm in size, which is 1-2 orders of magnitude smaller than existing technology. We present newly developed patterning and microfabrication methods for polymer microactuators as well as the latest results to integrate these microactuators into easy to use manipulation tools. The outcomes of this study contribute to the realisation of low-foot print devices articulated with electroactive polymer actuators for which the physical interface with the power source has been a significant challenge limiting their application. Here, we present a new bottom-up microfabrication process. We show for the first time that such a bottom-up fabricated actuator performs a movement in air. This is a significant step towards widening the application areas of the soft microactuators.

Green fluorescent protein nanopolygons as monodisperse supramolecular assemblies of functional proteins with defined valency

PubMed Central

Kim, Young Eun; Kim, Yu-na; Kim, Jung A.; Kim, Ho Min; Jung, Yongwon

2015-01-01

Supramolecular protein assemblies offer novel nanoscale architectures with molecular precision and unparalleled functional diversity. A key challenge, however, is to create precise nano-assemblies of functional proteins with both defined structures and a controlled number of protein-building blocks. Here we report a series of supramolecular green fluorescent protein oligomers that are assembled in precise polygonal geometries and prepared in a monodisperse population. Green fluorescent protein is engineered to be self-assembled in cells into oligomeric assemblies that are natively separated in a single-protein resolution by surface charge manipulation, affording monodisperse protein (nano)polygons from dimer to decamer. Several functional proteins are multivalently displayed on the oligomers with controlled orientations. Spatial arrangements of protein oligomers and displayed functional proteins are directly visualized by a transmission electron microscope. By employing our functional protein assemblies, we provide experimental insight into multivalent protein–protein interactions and tools to manipulate receptor clustering on live cell surfaces. PMID:25972078

Chromatibody, a novel non-invasive molecular tool to explore and manipulate chromatin in living cells

PubMed Central

Jullien, Denis; Vignard, Julien; Fedor, Yoann; Béry, Nicolas; Olichon, Aurélien; Crozatier, Michèle; Erard, Monique; Cassard, Hervé; Ducommun, Bernard; Salles, Bernard

2016-01-01

ABSTRACT Chromatin function is involved in many cellular processes, its visualization or modification being essential in many developmental or cellular studies. Here, we present the characterization of chromatibody, a chromatin-binding single-domain, and explore its use in living cells. This non-intercalating tool specifically binds the heterodimer of H2A–H2B histones and displays a versatile reactivity, specifically labeling chromatin from yeast to mammals. We show that this genetically encoded probe, when fused to fluorescent proteins, allows non-invasive real-time chromatin imaging. Chromatibody is a dynamic chromatin probe that can be modulated. Finally, chromatibody is an efficient tool to target an enzymatic activity to the nucleosome, such as the DNA damage-dependent H2A ubiquitylation, which can modify this epigenetic mark at the scale of the genome and result in DNA damage signaling and repair defects. Taken together, these results identify chromatibody as a universal non-invasive tool for either in vivo chromatin imaging or to manipulate the chromatin landscape. PMID:27206857

Piezoelectric translator. A simple and inexpensive device to move microelectrodes and micropipettes small distances rapidly.

PubMed

Lederer, W J

1983-09-01

A device is described that is capable of rapidly moving microelectrodes and micropipettes over distances up to 15 mu. This piezoelectric transLator uses the diaphragm from virtually any available piezoelectric buzzer in combination with simple physical support and drive electronics. All of the necessary details for the construction of this small device are presented. Each finished unit is about 2 cm long with a diameter of 2 cm and can be readily adapted to existing manipulators. The translator has been found useful in aiding the independent penetration by one or more microelectrodes of single cells or of more complicated multicellular preparations (including those that lie behind a connective tissue layer). This new device offers fine control of microelectrode motion that cannot be obtained by the other methods used to aid microelectrode and micropipette penetration of cell membranes (e.g. capacitance overcompensation--"ringing in"' or "tickling"--or tapping the manipulator base). Finally, the device described in this paper is extremely simple and inexpensive to build.

An integrated cell culture lab on a chip: modular microdevices for cultivation of mammalian cells and delivery into microfluidic microdroplets.

PubMed

Hufnagel, Hansjörg; Huebner, Ansgar; Gülch, Carina; Güse, Katharina; Abell, Chris; Hollfelder, Florian

2009-06-07

We present a modular system of microfluidic PDMS devices designed to incorporate the steps necessary for cell biological assays based on mammalian tissue culture 'on-chip'. The methods described herein include the on-chip immobilization and culturing of cells as well as their manipulation by transfection. Assessment of cell viability by flow cytrometry suggests low attrition rates (<3%) and excellent growth properties in the device for up to 7 days for CHO-K1 cells. To demonstrate that key procedures from the repertoire of cell biology are possible in this format, transfection of a reporter gene (encoding green fluorescent protein) was carried out. The modular design enables efficient detachment and recollection of cells and allows assessment of the success of transfection achieved on-chip. The transfection levels (20%) are comparable to standard large scale procedures and more than 500 cells could be transfected. Finally, cells are transferred into microfluidic microdoplets, where in principle a wide range of subsequent assays can be carried out at the single cell level in droplet compartments. The procedures developed for this modular device layout further demonstrate that commonly used methods in cell biology involving mammalian cells can be reliably scaled down to allow single cell investigations in picolitre volumes.

Modeling and control for micro and nano manipulation

NASA Astrophysics Data System (ADS)

Wejinya, Uchechukwu C.

Manipulation of micro and nano entities implies the movement of micro and nano entities from an initial position (location) to the desire position (location). This operation is not only necessary, but a required task with great precision. The tools needed for the manipulation needs to be chosen properly because the capabilities of the human hand are very restricted. Smart micro and nano manipulation are becoming of great interest in many applications including medicine and industry. In industry, high precision manipulation systems are especially needed for mass production of both micro and nano systems which consist of different component in respective scales. The transition from assembling and manipulating micro and nano entities manually to mass products with high quality is only attainable by automated assembly and manipulation systems. An example is the testing of integrated circuits which can be carried out by exchanging the manipulation tool by an electric probe. Furthermore, in medical research it is customary to pick up a single cell (human, plant, or animal), and carry it to another device which is used to further analyze the cell. Consequently, the cell of interest has to be separated from the other cells and picked up using the appropriate micro/nano tool. Hence it becomes absolutely necessary that the appropriate tool be used for specific micro or nano entity manipulation and assembly. In this research, we focus on developing micro tool for manipulating micro and nano entities in liquid environment using a micro fluidic end effector system with in-situ Polyvinylidene Fluoride (PVDF) sensing. The microfluidic end effector system consists of a DC micro-diaphragm pump and compressor, one region of flexible latex tube, a Polyvinylidene Fluoride (PVDF) sensor for in-situ measurement of micro drag force, and a micro pipette. The micro pipette of the novel microfluidic end effector system has an internal diameter (ID) smaller than 20mum used for microfluidic handling and deposition of micro and nano entities such as carbon nanotubes (CNT), DNA, and for droplet control. The novel microfluidic end effector system with force sensing can significantly improve the success rate for handling/depositing micro/nano entities in the case of carbon nanotubes between micro electrodes. The design, calibration, and experimental implementation of the novel microfluidic end effector is carried out in this research. The experimental results show the success rate for carbon nanotube(s) deposition between micro electrodes can reach close to 80%. Furthermore, carbon nanotubes are of particular interest because they are good candidates for many electronics and sensing applications. The interests in using carbon nanotubes to manufacture electronics and sensors have increased in recent years because of the increase need for making electronics smaller, and their excellent electrical and mechanical properties. These potentials can be achieved if CNTs with semiconducting and metallic band structure can be successfully deposited and separated. The use of dielectrophoresis (DEP) has been established as a course towards the efficient deposition and separation of metallic carbon nanotubes from semiconducting carbon nanotubes. For this reason, this research presents a new mathematical model for dielectrophoresis and electrorotation of carbon nanotubes. Simulation results are presented in this research to validate the developed model. The combination of both the micro robotic manipulation system and the atomic force microscopy (AFM) based nano-robotic system will provide a powerful tool for micro and nano manipulation. Additional applications of this research are endless considering the rapid development of micro and nano technologies.

Real-time detection and monitoring of the drug resistance of single myeloid leukemia cells by diffused total internal reflection.

PubMed

Liang, L; Jin, Y X; Zhu, X Q; Zhou, F L; Yang, Y

2018-05-15

Real-time detection and monitoring of the drug resistance of single cells have important significance in clinical diagnosis and therapy. Traditional methods operate a number of times for each individual concentration, and innovation is required for the design of more simple and efficient manipulation platforms with necessary higher sensitivity. Here, we have developed a novel diffused total internal reflection (TIR) method to perform drug metabolism and cytotoxicity analysis of trapped myeloid leukemia cells. Molm-13 cells, a type of acute myeloid leukemia cell, were chosen and injected into the device and fittingly captured by cell traps. Differing from previous studies, a series of different concentrations of azelaic acid (AZA) drug could be used from 0 mM to 50 mM through convection and diffusion processes in a single chip, with each concentration region featuring 50 cells, with a total of 549 cell trapping units. Thanks to the high sensitivity of the TIR method, only cells with the same drug concentration could be illuminated in the detection process. By adjusting the incident angle, we could exactly detect and monitor the drug resistance of the cells using different drug concentrations and the experimental resolution of the drug concentration was as small as 5 mM. Images of the membrane integrity and morphology of the cells in the bright field were measured and we also monitored the cell viabilities in the dark field over 2 hours. The effects of AZA on the Molm-13 cells were explored in different concentrations at the single cell level. Compared with the results of the traditional MTT assay method, the experimental results are more simple and accurate. A cell death of 5% at an AZA concentration of 5 mM was observed after 30 minutes, while a concentration of 40 mM corresponded to a 98% cell death. The designed method in this study provides a novel toolkit to control and monitor drug resistance at the single cell level more easily with higher sensitivity and we believe it has significant potential application in single cell quality assessment and medicine analysis in clinical practice.

Comprehensive Analysis of Transcription Dynamics from Brain Samples Following Behavioral Experience

PubMed Central

Turm, Hagit; Mukherjee, Diptendu; Haritan, Doron; Tahor, Maayan; Citri, Ami

2014-01-01

The encoding of experiences in the brain and the consolidation of long-term memories depend on gene transcription. Identifying the function of specific genes in encoding experience is one of the main objectives of molecular neuroscience. Furthermore, the functional association of defined genes with specific behaviors has implications for understanding the basis of neuropsychiatric disorders. Induction of robust transcription programs has been observed in the brains of mice following various behavioral manipulations. While some genetic elements are utilized recurrently following different behavioral manipulations and in different brain nuclei, transcriptional programs are overall unique to the inducing stimuli and the structure in which they are studied1,2. In this publication, a protocol is described for robust and comprehensive transcriptional profiling from brain nuclei of mice in response to behavioral manipulation. The protocol is demonstrated in the context of analysis of gene expression dynamics in the nucleus accumbens following acute cocaine experience. Subsequent to a defined in vivo experience, the target neural tissue is dissected; followed by RNA purification, reverse transcription and utilization of microfluidic arrays for comprehensive qPCR analysis of multiple target genes. This protocol is geared towards comprehensive analysis (addressing 50-500 genes) of limiting quantities of starting material, such as small brain samples or even single cells. The protocol is most advantageous for parallel analysis of multiple samples (e.g. single cells, dynamic analysis following pharmaceutical, viral or behavioral perturbations). However, the protocol could also serve for the characterization and quality assurance of samples prior to whole-genome studies by microarrays or RNAseq, as well as validation of data obtained from whole-genome studies. PMID:25225819

Atomic force microscopy and spectroscopy to probe single membrane proteins in lipid bilayers.

PubMed

Sapra, K Tanuj

2013-01-01

The atomic force microscope (AFM) has opened vast avenues hitherto inaccessible to the biological scientist. The high temporal (millisecond) and spatial (nanometer) resolutions of the AFM are suited for studying many biological processes in their native conditions. The AFM cantilever stylus is aptly termed as a "lab on a tip" owing to its versatility as an imaging tool as well as a handle to manipulate single bonds and proteins. Recent examples assert that the AFM can be used to study the mechanical properties and monitor processes of single proteins and single cells, thus affording insight into important mechanistic details. This chapter specifically focuses on practical and analytical protocols of single-molecule AFM methodologies related to high-resolution imaging and single-molecule force spectroscopy of membrane proteins. Both these techniques are operator oriented, and require specialized working knowledge of the instrument, theoretical, and practical skills.

Mechanical diagnosis of human erythrocytes by ultra-high speed manipulation unraveled critical time window for global cytoskeletal remodeling

NASA Astrophysics Data System (ADS)

Ito, Hiroaki; Murakami, Ryo; Sakuma, Shinya; Tsai, Chia-Hung Dylan; Gutsmann, Thomas; Brandenburg, Klaus; Pöschl, Johannes M. B.; Arai, Fumihito; Kaneko, Makoto; Tanaka, Motomu

2017-02-01

Large deformability of erythrocytes in microvasculature is a prerequisite to realize smooth circulation. We develop a novel tool for the three-step “Catch-Load-Launch” manipulation of a human erythrocyte based on an ultra-high speed position control by a microfluidic “robotic pump”. Quantification of the erythrocyte shape recovery as a function of loading time uncovered the critical time window for the transition between fast and slow recoveries. The comparison with erythrocytes under depletion of adenosine triphosphate revealed that the cytoskeletal remodeling over a whole cell occurs in 3 orders of magnitude longer timescale than the local dissociation-reassociation of a single spectrin node. Finally, we modeled septic conditions by incubating erythrocytes with endotoxin, and found that the exposure to endotoxin results in a significant delay in the characteristic transition time for cytoskeletal remodeling. The high speed manipulation of erythrocytes with a robotic pump technique allows for high throughput mechanical diagnosis of blood-related diseases.

In Vitro Electrochemistry of Biological Systems

PubMed Central

Adams, Kelly L.; Puchades, Maja; Ewing, Andrew G.

2009-01-01

This article reviews recent work involving electrochemical methods for in vitro analysis of biomolecules, with an emphasis on detection and manipulation at and of single cells and cultures of cells. The techniques discussed include constant potential amperometry, chronoamperometry, cellular electroporation, scanning electrochemical microscopy, and microfluidic platforms integrated with electrochemical detection. The principles of these methods are briefly described, followed in most cases with a short description of an analytical or biological application and its significance. The use of electrochemical methods to examine specific mechanistic issues in exocytosis is highlighted, as a great deal of recent work has been devoted to this application. PMID:20151038

Cooling assembly for fuel cells

DOEpatents

Kaufman, Arthur; Werth, John

1990-01-01

A cooling assembly for fuel cells having a simplified construction whereby coolant is efficiently circulated through a conduit arranged in serpentine fashion in a channel within a member of such assembly. The channel is adapted to cradle a flexible, chemically inert, conformable conduit capable of manipulation into a variety of cooling patterns without crimping or otherwise restricting of coolant flow. The conduit, when assembled with the member, conforms into intimate contact with the member for good thermal conductivity. The conduit is non-corrodible and can be constructed as a single, manifold-free, continuous coolant passage means having only one inlet and one outlet.

In vivo acoustic and photoacoustic focusing of circulating cells

NASA Astrophysics Data System (ADS)

Galanzha, Ekaterina I.; Viegas, Mark G.; Malinsky, Taras I.; Melerzanov, Alexander V.; Juratli, Mazen A.; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Zharov, Vladimir P.

2016-03-01

In vivo flow cytometry using vessels as natural tubes with native cell flows has revolutionized the study of rare circulating tumor cells in a complex blood background. However, the presence of many blood cells in the detection volume makes it difficult to count each cell in this volume. We introduce method for manipulation of circulating cells in vivo with the use of gradient acoustic forces induced by ultrasound and photoacoustic waves. In a murine model, we demonstrated cell trapping, redirecting and focusing in blood and lymph flow into a tight stream, noninvasive wall-free transportation of blood, and the potential for photoacoustic detection of sickle cells without labeling and of leukocytes targeted by functionalized nanoparticles. Integration of cell focusing with intravital imaging methods may provide a versatile biological tool for single-cell analysis in circulation, with a focus on in vivo needleless blood tests, and preclinical studies of human diseases in animal models.

In vivo acoustic and photoacoustic focusing of circulating cells

PubMed Central

Galanzha, Ekaterina I.; Viegas, Mark G.; Malinsky, Taras I.; Melerzanov, Alexander V.; Juratli, Mazen A.; Sarimollaoglu, Mustafa; Nedosekin, Dmitry A.; Zharov, Vladimir P.

2016-01-01

In vivo flow cytometry using vessels as natural tubes with native cell flows has revolutionized the study of rare circulating tumor cells in a complex blood background. However, the presence of many blood cells in the detection volume makes it difficult to count each cell in this volume. We introduce method for manipulation of circulating cells in vivo with the use of gradient acoustic forces induced by ultrasound and photoacoustic waves. In a murine model, we demonstrated cell trapping, redirecting and focusing in blood and lymph flow into a tight stream, noninvasive wall-free transportation of blood, and the potential for photoacoustic detection of sickle cells without labeling and of leukocytes targeted by functionalized nanoparticles. Integration of cell focusing with intravital imaging methods may provide a versatile biological tool for single-cell analysis in circulation, with a focus on in vivo needleless blood tests, and preclinical studies of human diseases in animal models. PMID:26979811

Zscan4 restores the developmental potency of embryonic stem cells

PubMed Central

Amano, Tomokazu; Hirata, Tetsuya; Falco, Geppino; Monti, Manuela; Sharova, Lioudmila V.; Amano, Misa; Sheer, Sarah; Hoang, Hien G.; Piao, Yulan; Stagg, Carole A.; Yamamizu, Kohei; Akiyama, Tomohiko; Ko, Minoru S.H.

2013-01-01

The developmental potency of mouse embryonic stem (ES) cells, which is the ability to contribute to a whole embryo is known to deteriorate during long-term cell culture. Previously we have shown that ES cells oscillate between Zscan4- and Zscan4+ states, and the transient activation of Zscan4 is required for the maintenance of telomeres and genome stability of ES cells. Here we show that increasing the frequency of Zscan4 activation in mouse ES cells restores and maintains their developmental potency in long-term cell culture. Injection of a single ES cell with such increased potency into a tetraploid blastocyst gives rise to an entire embryo with a higher success rate. These results not only provide a means to rejuvenate ES cells by manipulating Zscan4 expression, but also indicate the active roles of Zscan4 in the long-term maintenance of ES cell potency. PMID:23739662

Design of a novel magnetic platform for cell manipulation

NASA Astrophysics Data System (ADS)

Lucarini, Gioia; Iacovacci, Veronica; Gouveia, Pedro J.; Ricotti, Leonardo; Menciassi, Arianna

2018-02-01

Cell manipulation tasks, especially in lab-on-a-chip applications for personalized medicine, could greatly benefit from mobile untethered microdevices able to wirelessly navigate in fluidic environments by means of magnetic fields. In this paper, the design, fabrication and testing of a magnetic platform enabling the controlled locomotion and immersion of microrobots placed at the air/liquid interface is proposed and exploited for cell manipulation. The proposed microrobot consists of a polymeric magnetic thin film that acts as cell transporter and a specific coating strategy, devised to enhance a safe cancer cell adhesion to the magnetic film. Experimental results demonstrated an overall cell viability and a fine control of magnetic microrobot locomotion. The proposed technologies are promising in view of future cell manipulation tasks for personalized medicine applications.

Polarization manipulation in single refractive prism based holography lithography

NASA Astrophysics Data System (ADS)

Xiong, Wenjie; Xu, Yi; Xiao, Yujian; Lv, Xiaoxu; Wu, Lijun

2015-01-01

We propose theoretically and demonstrate experimentally a simple but effective strategy for polarization manipulation in single refractive prism based holographic lithography. By tuning the polarization of a single laser beam, we can obtain the pill shape interference pattern with a high-contrast where a complex optical setup and multiple polarizers are needed in the conventional holography lithography. Fabrication of pill shape two-dimensional polymer photonic crystals using one beam and one shoot holography lithography is shown as an example to support our theoretical results. This integrated polarization manipulation technique can release the crucial stability restrictions imposed on the multiple beams holography lithography.

CAVITATION SOUNDS DURING CERVICOTHORACIC SPINAL MANIPULATION.

PubMed

Dunning, James; Mourad, Firas; Zingoni, Andrea; Iorio, Raffaele; Perreault, Thomas; Zacharko, Noah; de Las Peñas, César Fernández; Butts, Raymond; Cleland, Joshua A

2017-08-01

No study has previously investigated the side, duration or number of audible cavitation sounds during high-velocity low-amplitude (HVLA) thrust manipulation to the cervicothoracic spine. The primary purpose was to determine which side of the spine cavitates during cervicothoracic junction (CTJ) HVLA thrust manipulation. Secondary aims were to calculate the average number of cavitations, the duration of cervicothoracic thrust manipulation, and the duration of a single cavitation. Quasi-experimental study. Thirty-two patients with upper trapezius myalgia received two cervicothoracic HVLA thrust manipulations targeting the right and left T1-2 articulation, respectively. Two high sampling rate accelerometers were secured bilaterally 25 mm lateral to midline of the T1-2 interspace. For each manipulation, two audio signals were extracted using Short-Time Fourier Transformation (STFT) and singularly processed via spectrogram calculation in order to evaluate the frequency content and number of instantaneous energy bursts of both signals over time for each side of the CTJ. Unilateral cavitation sounds were detected in 53 (91.4%) of 58 cervicothoracic HVLA thrust manipulations and bilateral cavitation sounds were detected in just five (8.6%) of the 58 thrust manipulations; that is, cavitation was significantly (p<0.001) more likely to occur unilaterally than bilaterally. In addition, cavitation was significantly (p<0.0001) more likely to occur on the side contralateral to the clinician's short-lever applicator. The mean number of audible cavitations per manipulation was 4.35 (95% CI 2.88, 5.76). The mean duration of a single manipulation was 60.77 ms (95% CI 28.25, 97.42) and the mean duration of a single audible cavitation was 4.13 ms (95% CI 0.82, 7.46). In addition to single-peak and multi-peak energy bursts, spectrogram analysis also demonstrated high frequency sounds, low frequency sounds, and sounds of multiple frequencies for all 58 manipulations. Cavitation was significantly more likely to occur unilaterally, and on the side contralateral to the short-lever applicator contact, during cervicothoracic HVLA thrust manipulation. Clinicians should expect multiple cavitation sounds when performing HVLA thrust manipulation to the CTJ. Due to the presence of multi-peak energy bursts and sounds of multiple frequencies, the cavitation hypothesis (i.e. intra-articular gas bubble collapse) alone appears unable to explain all of the audible sounds during HVLA thrust manipulation, and the possibility remains that several phenomena may be occurring simultaneously. 2b.

Acute effects of single and multiple level thoracic manipulations on chronic mechanical neck pain: a randomized controlled trial

PubMed Central

Puntumetakul, Rungthip; Suvarnnato, Thavatchai; Werasirirat, Phurichaya; Uthaikhup, Sureeporn; Yamauchi, Junichiro; Boucaut, Rose

2015-01-01

Background Thoracic spine manipulation has become a popular alternative to local cervical manipulative therapy for mechanical neck pain. This study investigated the acute effects of single-level and multiple-level thoracic manipulations on chronic mechanical neck pain (CMNP). Methods Forty-eight patients with CMNP were randomly allocated to single-level thoracic manipulation (STM) at T6–T7 or multiple-level thoracic manipulation (MTM), or to a control group (prone lying). Cervical range of motion (CROM), visual analog scale (VAS), and the Thai version of the Neck Disability Index (NDI-TH) scores were measured at baseline, and at 24-hour and at 1-week follow-up. Results At 24-hour and 1-week follow-up, neck disability and pain levels were significantly (P<0.05) improved in the STM and MTM groups compared with the control group. CROM in flexion and left lateral flexion were increased significantly (P<0.05) in the STM group when compared with the control group at 1-week follow-up. The CROM in right rotation was increased significantly after MTM compared to the control group (P<0.05) at 24-hour follow-up. There were no statistically significant differences in neck disability, pain level at rest, and CROM between the STM and MTM groups. Conclusion These results suggest that both single-level and multiple-level thoracic manipulation improve neck disability, pain levels, and CROM at 24-hour and 1-week follow-up in patients with CMNP. PMID:25624764

Displacement correlations between a single mesenchymal-like cell and its nucleus effectively link subcellular activities and motility in cell migration analysis

NASA Astrophysics Data System (ADS)

Lan, Tian; Cheng, Kai; Ren, Tina; Arce, Stephen Hugo; Tseng, Yiider

2016-09-01

Cell migration is an essential process in organism development and physiological maintenance. Although current methods permit accurate comparisons of the effects of molecular manipulations and drug applications on cell motility, effects of alterations in subcellular activities on motility cannot be fully elucidated from those methods. Here, we develop a strategy termed cell-nuclear (CN) correlation to parameterize represented dynamic subcellular activities and to quantify their contributions in mesenchymal-like migration. Based on the biophysical meaning of the CN correlation, we propose a cell migration potential index (CMPI) to measure cell motility. When the effectiveness of CMPI was evaluated with respect to one of the most popular cell migration analysis methods, Persistent Random Walk, we found that the cell motility estimates among six cell lines used in this study were highly consistent between these two approaches. Further evaluations indicated that CMPI can be determined using a shorter time period and smaller cell sample size, and it possesses excellent reliability and applicability, even in the presence of a wide range of noise, as might be generated from individual imaging acquisition systems. The novel approach outlined here introduces a robust strategy through an analysis of subcellular locomotion activities for single cell migration assessment.

Parallel manipulation of individual magnetic microbeads for lab-on-a-chip applications

NASA Astrophysics Data System (ADS)

Peng, Zhengchun

Many scientists and engineers are turning to lab-on-a-chip systems for faster and cheaper analysis of chemical reactions and biomolecular interactions. A common approach that facilitates the handling of reagents and biomolecules in these systems utilizes micro/nano beads as the solid carrier. Physical manipulation, such as assembly, transport, sorting, and tweezing, of beads on a chip represents an essential step for fully utilizing their potentials in a wide spectrum of bead-based analysis. Previous work demonstrated manipulation of either an ensemble of beads without individual control, or single beads but lacks the capability for parallel operation. Parallel manipulation of individual beads is required to meet the demand for high-throughput and location-specific analysis. In this work, we introduced two methods for parallel manipulation of individual magnetic microbeads, which can serve as effective lab-on-a-chip platforms and/or efficient analytic tools. The first method employs arrays of soft ferromagnetic patterns fabricated inside a microfluidic channel and subjected to an external magnetic field. We demonstrated that the system can be used to assemble individual beads (1-3 mum) from a flow of suspended beads into a regular array on the chip, hence improving the integrated electrochemical detection of biomolecules bound to the bead surface. By rotating the external field, the assembled microbeads can be remotely controlled with synchronized, high-speed circular motion around individual soft magnets on the chip. We employed this manipulation mode for efficient sample mixing in continuous microflow. Furthermore, we discovered a simple but effective way of transporting the microbeads on the chip by varying the strength of the local bias field within a revolution of the external field. In addition, selective transport of microbeads with different size was realized, providing a platform for effective on-chip sample separation and offering the potential for multiplexing capability. The second method integrates magnetic and dielectrophoretic manipulations of the same microbeads. The device combines tapered conducting wires and fingered electrodes to generate desirable magnetic and electric fields, respectively. By externally programming the magnetic attraction and dielectrophoretic repulsion forces, out-of-plane oscillation of the microbeads across the channel height was realized. This manipulation mode can facilitate the interaction between the beads with multiple layers of sample fluid inside the channel. We further demonstrated the tweezing of microbeads in liquid with high spatial resolutions, i.e., from submicrometer to nanometer range, by fine-tuning the net force from magnetic attraction and dielectrophoretic repulsion of the beads. The highresolution control of the out-of-plane motion of the microbeads led to the invention of massively parallel biomolecular tweezers. We believe the maturation of bead-based microtweezers will revolutionize the state-of-art tools currently used for single cell and single molecule studies.

Methodological challenges of optical tweezers-based X-ray fluorescence imaging of biological model organisms at synchrotron facilities.

PubMed

Vergucht, Eva; Brans, Toon; Beunis, Filip; Garrevoet, Jan; Bauters, Stephen; De Rijcke, Maarten; Deruytter, David; Janssen, Colin; Riekel, Christian; Burghammer, Manfred; Vincze, Laszlo

2015-07-01

Recently, a radically new synchrotron radiation-based elemental imaging approach for the analysis of biological model organisms and single cells in their natural in vivo state was introduced. The methodology combines optical tweezers (OT) technology for non-contact laser-based sample manipulation with synchrotron radiation confocal X-ray fluorescence (XRF) microimaging for the first time at ESRF-ID13. The optical manipulation possibilities and limitations of biological model organisms, the OT setup developments for XRF imaging and the confocal XRF-related challenges are reported. In general, the applicability of the OT-based setup is extended with the aim of introducing the OT XRF methodology in all research fields where highly sensitive in vivo multi-elemental analysis is of relevance at the (sub)micrometre spatial resolution level.

Virtual reality visual feedback for hand-controlled scanning probe microscopy manipulation of single molecules.

PubMed

Leinen, Philipp; Green, Matthew F B; Esat, Taner; Wagner, Christian; Tautz, F Stefan; Temirov, Ruslan

2015-01-01

Controlled manipulation of single molecules is an important step towards the fabrication of single molecule devices and nanoscale molecular machines. Currently, scanning probe microscopy (SPM) is the only technique that facilitates direct imaging and manipulations of nanometer-sized molecular compounds on surfaces. The technique of hand-controlled manipulation (HCM) introduced recently in Beilstein J. Nanotechnol. 2014, 5, 1926-1932 simplifies the identification of successful manipulation protocols in situations when the interaction pattern of the manipulated molecule with its environment is not fully known. Here we present a further technical development that substantially improves the effectiveness of HCM. By adding Oculus Rift virtual reality goggles to our HCM set-up we provide the experimentalist with 3D visual feedback that displays the currently executed trajectory and the position of the SPM tip during manipulation in real time, while simultaneously plotting the experimentally measured frequency shift (Δf) of the non-contact atomic force microscope (NC-AFM) tuning fork sensor as well as the magnitude of the electric current (I) flowing between the tip and the surface. The advantages of the set-up are demonstrated by applying it to the model problem of the extraction of an individual PTCDA molecule from its hydrogen-bonded monolayer grown on Ag(111) surface.

Atomic force microscopy – looking at mechanosensors on the cell surface

PubMed Central

Heinisch, Jürgen J.; Lipke, Peter N.; Beaussart, Audrey; El Kirat Chatel, Sofiane; Dupres, Vincent; Alsteens, David; Dufrêne, Yves F.

2012-01-01

Summary Living cells use cell surface proteins, such as mechanosensors, to constantly sense and respond to their environment. However, the way in which these proteins respond to mechanical stimuli and assemble into large complexes remains poorly understood at the molecular level. In the past years, atomic force microscopy (AFM) has revolutionized the way in which biologists analyze cell surface proteins to molecular resolution. In this Commentary, we discuss how the powerful set of advanced AFM techniques (e.g. live-cell imaging and single-molecule manipulation) can be integrated with the modern tools of molecular genetics (i.e. protein design) to study the localization and molecular elasticity of individual mechanosensors on the surface of living cells. Although we emphasize recent studies on cell surface proteins from yeasts, the techniques described are applicable to surface proteins from virtually all organisms, from bacteria to human cells. PMID:23077172

Single cell Enrichment with High Throughput Microfluidic Devices

NASA Astrophysics Data System (ADS)

Pakjesm Pourfard, Pedram

Microfluidics is a rapidly growing field of biomedical engineering with numerous applications such as diagnostic testing, therapeutics, and research preparation. Cell enrichment for automated diagnostic is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as, Shear migration, Lift force, Dean force, and many other label-free techniques, are advantageous since they don't require costly labeling or sample preparation. However, current passive techniques for enrichment had limited adoption in clinical and cell biology research applications. They generally require low flow rate and low cell volume fraction for high efficiency. The Control increment filtration, T-shaped microfluidic device, and spiral-shaped microfluidic devices will be studied for single-cell separation from aggregates. Control increment filtration works like the tangential filter; however, cells are separated based off of same amount of flow rate passing through large space gaps. Main microchannel of T-Shaped is connected to two perpendicular side channels. Based off Shear-modulated inertial migration, this device will enable selective enrichment of cells. The spiral shaped microfluidic device depends on different Dean and lift forces acting on cells to separate them based off different sizes. The spiral geometry of the microchannel will enable dominant inertial forces and the Dean Rotation force to cause larger cells to migrate to the inner side of the microchannel. Because manipulation of microchannel dimensions correlates to the degree of cell separation, versatility in design exists. Cell mixture samples will contain cells of different sizes and therefore design strategies could be utilized to maximize the effectiveness of single-cell separation.

On the manipulability of dual cooperative robots

NASA Technical Reports Server (NTRS)

Chiacchio, P.; Chiaverini, S.; Sciavicco, L.; Siciliano, B.

1989-01-01

The definition of manipulability ellipsoids for dual robot systems is given. A suitable kineto-static formulation for dual cooperative robots is adopted which allows for a global task space description of external and internal forces, and relative velocities. The well known concepts of force and velocity manipulability ellipsoids for a single robot are formally extended and the contributions of the two single robots to the cooperative system ellipsoids are illustrated. Duality properties are discussed. A practical case study is developed.

A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces.

PubMed

Gross, Benjamin J; El-Naggar, Mohamed Y

2015-06-01

Metal-reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. This process is one of the earliest forms of respiration on earth and has significant environmental and technological implications. By performing electron transfer to electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. Understanding such a complex biotic-abiotic interaction necessitates the development of tools capable of probing extracellular electron transfer down to the level of single cells. Here, we describe an experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect the respiration current correlated with cell-electrode contact. We demonstrate the system with single cell measurements of the dissimilatory-metal reducing bacterium Shewanella oneidensis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer-membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. In addition to the application for extracellular electron transfer studies, the ability to electronically measure cell-specific respiration rates may provide answers for a variety of fundamental microbial physiology questions.

A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces

NASA Astrophysics Data System (ADS)

Gross, Benjamin J.; El-Naggar, Mohamed Y.

2015-06-01

Metal-reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. This process is one of the earliest forms of respiration on earth and has significant environmental and technological implications. By performing electron transfer to electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. Understanding such a complex biotic-abiotic interaction necessitates the development of tools capable of probing extracellular electron transfer down to the level of single cells. Here, we describe an experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect the respiration current correlated with cell-electrode contact. We demonstrate the system with single cell measurements of the dissimilatory-metal reducing bacterium Shewanella oneidensis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer-membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. In addition to the application for extracellular electron transfer studies, the ability to electronically measure cell-specific respiration rates may provide answers for a variety of fundamental microbial physiology questions.

Detectors for single-molecule fluorescence imaging and spectroscopy

PubMed Central

MICHALET, X.; SIEGMUND, O.H.W.; VALLERGA, J.V.; JELINSKY, P.; MILLAUD, J.E.; WEISS, S.

2010-01-01

Single-molecule observation, characterization and manipulation techniques have recently come to the forefront of several research domains spanning chemistry, biology and physics. Due to the exquisite sensitivity, specificity, and unmasking of ensemble averaging, single-molecule fluorescence imaging and spectroscopy have become, in a short period of time, important tools in cell biology, biochemistry and biophysics. These methods led to new ways of thinking about biological processes such as viral infection, receptor diffusion and oligomerization, cellular signaling, protein-protein or protein-nucleic acid interactions, and molecular machines. Such achievements require a combination of several factors to be met, among which detector sensitivity and bandwidth are crucial. We examine here the needed performance of photodetectors used in these types of experiments, the current state of the art for different categories of detectors, and actual and future developments of single-photon counting detectors for single-molecule imaging and spectroscopy. PMID:20157633

Laser Surgery: Organelles to Organs

NASA Astrophysics Data System (ADS)

Berns, Michael W. D.

1998-03-01

Understanding the physical mechanisms of light interaction with biological molecules and structure has resulted in the application of photons to a wide variety of biological and medical problems ranging from subcellular manipulation/surgery to the successful diagnosis and treatment of human disease. Mechanisms such as the generation and transfer of heat, light-driven chemistry (photochemistry), high peak power acoustic-mechanical effects, high photon-energy induced bond breaking, and optical induced forces through momentum transfer, are being utilized in single cells at the microscopic (submicron and micron) level as well as the macroscopic level in tissue and organs. At the subcellular level, focused laser microbeams (laser scissors and tweezers) are being used to cut and move chromosomes to study genetic function as well as to clone and sequence genes. The same laser technology is being used to manipulate a variety of cell organelles such as mitochondria, cell membranes, nucleoli, and mitochondria in order to study their functions in cell physiology. At the tissue level, lasers are being used to diagnose and treat malignancy in combination with light-activated drugs, to ablate cornea and other hard and soft tissue through ultraviolet photoablation, to selectively ablate structures within the skin under controlled heating/cooling conditions, and to differentiate normal from abnormal tissue using a variety of fluorescence detection and light scattering techniques.

Bio-inspired configurable multiscale extracellular matrix-like structures for functional alignment and guided orientation of cells.

PubMed

Bae, Won-Gyu; Kim, Jangho; Choung, Yun-Hoon; Chung, Yesol; Suh, Kahp Y; Pang, Changhyun; Chung, Jong Hoon; Jeong, Hoon Eui

2015-11-01

Inspired by the hierarchically organized protein fibers in extracellular matrix (ECM) as well as the physiological importance of multiscale topography, we developed a simple but robust method for the design and manipulation of precisely controllable multiscale hierarchical structures using capillary force lithography in combination with an original wrinkling technique. In this study, based on our proposed fabrication technology, we approached a conceptual platform that can mimic the hierarchically multiscale topographical and orientation cues of the ECM for controlling cell structure and function. We patterned the polyurethane acrylate-based nanotopography with various orientations on the microgrooves, which could provide multiscale topography signals of ECM to control single and multicellular morphology and orientation with precision. Using our platforms, we found that the structures and orientations of fibroblast cells were greatly influenced by the nanotopography, rather than the microtopography. We also proposed a new approach that enables the generation of native ECM having nanofibers in specific three-dimensional (3D) configurations by culturing fibroblast cells on the multiscale substrata. We suggest that our methodology could be used as efficient strategies for the design and manipulation of various functional platforms, including well-defined 3D tissue structures for advanced regenerative medicine applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Electrophysiological Recordings from Lobula Plate Tangential Cells in Drosophila.

PubMed

Mauss, Alex S; Borst, Alexander

2016-01-01

Drosophila has emerged as an important model organism for the study of the neural basis of behavior. Its main asset is the experimental accessibility of identified neurons by genetic manipulation and physiological recordings. Drosophila therefore offers the opportunity to reach an integrative understanding of the development and neural underpinnings of behavior at all processing stages, from sensing to motor control, in a single species. Here, we will provide an account of the procedures involved in recording the electrical potential of individual neurons in the visual system of adult Drosophila using the whole-cell patch-clamp method. To this end, animals are fixed to a holder and mounted below a recording chamber. The head capsule is cut open and the glial sheath covering the brain is ruptured by a combination of shearing and enzymatic digest. Neuronal somata are thus exposed and targeted by low-resistance patch electrodes. After formation of a high resistance seal, electrical access to the cell is gained by small current pulses and suction. Stable recordings of large neurons are feasible for >1 h and can be combined with controlled visual stimulation as well as genetic and pharmacological manipulation of upstream circuit elements to infer circuit function in great detail.

Localized, macromolecular transport for thin, adherent, single cells via an automated, single cell electroporation biomanipulator.

PubMed

Sakaki, Kelly; Esmaeilsabzali, Hadi; Massah, Shabnam; Prefontaine, Gratien G; Dechev, Nikolai; Burke, Robert D; Park, Edward J

2013-11-01

Single cell electroporation (SCE), via microcapillary, is an effective method for molecular, transmembrane transport used to gain insight on cell processes with minimal preparation. Although possessing great potential, SCE is difficult to execute and the technology spans broad fields within cell biology and engineering. The technical complexities, the focus and expertise demanded during manual operation, and the lack of an automated SCE platform limit the widespread use of this technique, thus the potential of SCE has not been realized. In this study, an automated biomanipulator for SCE is presented. Our system is capable of delivering molecules into the cytoplasm of extremely thin cellular features of adherent cells. The intent of the system is to abstract the technical challenges and exploit the accuracy and repeatability of automated instrumentation, leaving only the focus of the experimental design to the operator. Each sequence of SCE including cell and SCE site localization, tip-membrane contact detection, and SCE has been automated. Positions of low-contrast cells are localized and "SCE sites" for microcapillary tip placement are determined using machine vision. In addition, new milestones within automated cell manipulation have been achieved. The system described herein has the capability of automated SCE of "thin" cell features less than 10 μm in thickness. Finally, SCE events are anticipated using visual feedback, while monitoring fluorescing dye entering the cytoplasm of a cell. The execution is demonstrated by inserting a combination of a fluorescing dye and a reporter gene into NIH/3T3 fibroblast cells.

Design of a surgical robot with dynamic vision field control for Single Port Endoscopic Surgery.

PubMed

Kobayashi, Yo; Sekiguchi, Yuta; Tomono, Yu; Watanabe, Hiroki; Toyoda, Kazutaka; Konishi, Kozo; Tomikawa, Morimasa; Ieiri, Satoshi; Tanoue, Kazuo; Hashizume, Makoto; Fujie, Masaktsu G

2010-01-01

Recently, a robotic system was developed to assist Single Port Endoscopic Surgery (SPS). However, the existing system required a manual change of vision field, hindering the surgical task and increasing the degrees of freedom (DOFs) of the manipulator. We proposed a surgical robot for SPS with dynamic vision field control, the endoscope view being manipulated by a master controller. The prototype robot consisted of a positioning and sheath manipulator (6 DOF) for vision field control, and dual tool tissue manipulators (gripping: 5DOF, cautery: 3DOF). Feasibility of the robot was demonstrated in vitro. The "cut and vision field control" (using tool manipulators) is suitable for precise cutting tasks in risky areas while a "cut by vision field control" (using a vision field control manipulator) is effective for rapid macro cutting of tissues. A resection task was accomplished using a combination of both methods.

Rapid determination of cell mass and density using digitally controlled electric field in a microfluidic chip.

PubMed

Zhao, Yuliang; Lai, Hok Sum Sam; Zhang, Guanglie; Lee, Gwo-Bin; Li, Wen Jung

2014-11-21

The density of a single cell is a fundamental property of cells. Cells in the same cycle phase have similar volume, but the differences in their mass and density could elucidate each cell's physiological state. Here we report a novel technique to rapidly measure the density and mass of a single cell using an optically induced electrokinetics (OEK) microfluidic platform. Presently, single cellular mass and density measurement devices require a complicated fabrication process and their output is not scalable, i.e., it is extremely difficult to measure the mass and density of a large quantity of cells rapidly. The technique reported here operates on a principle combining sedimentation theory, computer vision, and microparticle manipulation techniques in an OEK microfluidic platform. We will show in this paper that this technique enables the measurement of single-cell volume, density, and mass rapidly and accurately in a repeatable manner. The technique is also scalable - it allows simultaneous measurement of volume, density, and mass of multiple cells. Essentially, a simple time-controlled projected light pattern is used to illuminate the selected area on the OEK microfluidic chip that contains cells to lift the cells to a particular height above the chip's surface. Then, the cells are allowed to "free fall" to the chip's surface, with competing buoyancy, gravitational, and fluidic drag forces acting on the cells. By using a computer vision algorithm to accurately track the motion of the cells and then relate the cells' motion trajectory to sedimentation theory, the volume, mass, and density of each cell can be rapidly determined. A theoretical model of micro-sized spheres settling towards an infinite plane in a microfluidic environment is first derived and validated experimentally using standard micropolystyrene beads to demonstrate the viability and accuracy of this new technique. Next, we show that the yeast cell volume, mass, and density could be rapidly determined using this technology, with results comparable to those using the existing method suspended microchannel resonator.

Genetic strategies to investigate neuronal circuit properties using stem cell-derived neurons

PubMed Central

Garcia, Isabella; Kim, Cynthia; Arenkiel, Benjamin R.

2012-01-01

The mammalian brain is anatomically and functionally complex, and prone to diverse forms of injury and neuropathology. Scientists have long strived to develop cell replacement therapies to repair damaged and diseased nervous tissue. However, this goal has remained unrealized for various reasons, including nascent knowledge of neuronal development, the inability to track and manipulate transplanted cells within complex neuronal networks, and host graft rejection. Recent advances in embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) technology, alongside novel genetic strategies to mark and manipulate stem cell-derived neurons, now provide unprecedented opportunities to investigate complex neuronal circuits in both healthy and diseased brains. Here, we review current technologies aimed at generating and manipulating neurons derived from ESCs and iPSCs toward investigation and manipulation of complex neuronal circuits, ultimately leading to the design and development of novel cell-based therapeutic approaches. PMID:23264761

Virtual and concrete manipulatives: a comparison of approaches for solving mathematics problems for students with autism spectrum disorder.

PubMed

Bouck, Emily C; Satsangi, Rajiv; Doughty, Teresa Taber; Courtney, William T

2014-01-01

Students with autism spectrum disorder (ASD) are included in general education classes and expected to participate in general education content, such as mathematics. Yet, little research explores academically-based mathematics instruction for this population. This single subject alternating treatment design study explored the effectiveness of concrete (physical objects that can be manipulated) and virtual (3-D objects from the Internet that can be manipulated) manipulatives to teach single- and double-digit subtraction skills. Participants in this study included three elementary-aged students (ages ranging from 6 to 10) diagnosed with ASD. Students were selected from a clinic-based setting, where all participants received medically necessary intensive services provided via one-to-one, trained therapists. Both forms of manipulatives successfully assisted students in accurately and independently solving subtraction problem. However, all three students demonstrated greater accuracy and faster independence with the virtual manipulatives as compared to the concrete manipulatives. Beyond correctly solving the subtraction problems, students were also able to generalize their learning of subtraction through concrete and virtual manipulatives to more real-world applications.

Dual targeting of gene delivery by genetic modification of adenovirus serotype 5 fibers and cell-selective transcriptional control.

PubMed

Work, L M; Ritchie, N; Nicklin, S A; Reynolds, P N; Baker, A H

2004-08-01

Adenovirus (Ad)-mediated gene delivery is a promising approach for genetic manipulation of the vasculature and is being used in both preclinical models and clinical trials. However, safety concerns relating to infection of nontarget tissue and the poor infectivity of vascular cells compared to other cell types necessitates Ad vector refinement. Here, we combine a transductional targeting approach to improve vascular cell infectivity through RGD peptide insertion into adenovirus fibers, combined with transcriptional targeting to endothelial cells using a approximately 1 kb fragment of the fms-like tyrosine kinase receptor-1 (FLT-1) promoter. Single- and double-modified vectors were characterized in human cell lines that either support or have silenced FLT-1 expression. In rat hepatocytes and endothelial cells, the double modification substantially shifted transduction profiles toward vascular endothelial cells. Furthermore, in intact aortae derived from spontaneously hypertensive rats that display enhanced alphav integrin expression on dysfunctional endothelium, enhanced levels of transduction were observed using the double-modified vector but not in aortae derived from normotensive control rats. Our data indicate that Ad-mediated transduction can be beneficially modified in vitro and in vivo by combining fiber modification and a cell-selective promoter within a single-component vector system.

Manual and Instrument Applied Cervical Manipulation for Mechanical Neck Pain: A Randomized Controlled Trial.

PubMed

Gorrell, Lindsay M; Beath, Kenneth; Engel, Roger M

2016-06-01

The purpose of this study was to compare the effects of 2 different cervical manipulation techniques for mechanical neck pain (MNP). Participants with MNP of at least 1 month's duration (n = 65) were randomly allocated to 3 groups: (1) stretching (control), (2) stretching plus manually applied manipulation (MAM), and (3) stretching plus instrument-applied manipulation (IAM). MAM consisted of a single high-velocity, low-amplitude cervical chiropractic manipulation, whereas IAM involved the application of a single cervical manipulation using an (Activator IV) adjusting instrument. Preintervention and postintervention measurements were taken of all outcomes measures. Pain was the primary outcome and was measured using visual analogue scale and pressure pain thresholds. Secondary outcomes included cervical range of motion, hand grip-strength, and wrist blood pressure. Follow-up subjective pain scores were obtained via telephone text message 7 days postintervention. Subjective pain scores decreased at 7-day follow-up in the MAM group compared with control (P = .015). Cervical rotation bilaterally (ipsilateral: P = .002; contralateral: P = .015) and lateral flexion on the contralateral side to manipulation (P = .001) increased following MAM. Hand grip-strength on the contralateral side to manipulation (P = .013) increased following IAM. No moderate or severe adverse events were reported. Mild adverse events were reported on 6 occasions (control, 4; MAM, 1; IAM, 1). This study demonstrates that a single cervical manipulation is capable of producing immediate and short-term benefits for MNP. The study also demonstrates that not all manipulative techniques have the same effect and that the differences may be mediated by neurological or biomechanical factors inherent to each technique. Copyright © 2016. Published by Elsevier Inc.

Single cell adhesion assay using computer controlled micropipette.

PubMed

Salánki, Rita; Hős, Csaba; Orgovan, Norbert; Péter, Beatrix; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

2014-01-01

Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today's techniques typically have an extremely low throughput (5-10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of strongly fibrinogen adherent cells appearing in macrophages and highly represented in dendritic cells, but not observed in monocytes.

Single Cell Adhesion Assay Using Computer Controlled Micropipette

PubMed Central

Salánki, Rita; Hős, Csaba; Orgovan, Norbert; Péter, Beatrix; Sándor, Noémi; Bajtay, Zsuzsa; Erdei, Anna; Horvath, Robert; Szabó, Bálint

2014-01-01

Cell adhesion is a fundamental phenomenon vital for all multicellular organisms. Recognition of and adhesion to specific macromolecules is a crucial task of leukocytes to initiate the immune response. To gain statistically reliable information of cell adhesion, large numbers of cells should be measured. However, direct measurement of the adhesion force of single cells is still challenging and today’s techniques typically have an extremely low throughput (5–10 cells per day). Here, we introduce a computer controlled micropipette mounted onto a normal inverted microscope for probing single cell interactions with specific macromolecules. We calculated the estimated hydrodynamic lifting force acting on target cells by the numerical simulation of the flow at the micropipette tip. The adhesion force of surface attached cells could be accurately probed by repeating the pick-up process with increasing vacuum applied in the pipette positioned above the cell under investigation. Using the introduced methodology hundreds of cells adhered to specific macromolecules were measured one by one in a relatively short period of time (∼30 min). We blocked nonspecific cell adhesion by the protein non-adhesive PLL-g-PEG polymer. We found that human primary monocytes are less adherent to fibrinogen than their in vitro differentiated descendants: macrophages and dendritic cells, the latter producing the highest average adhesion force. Validation of the here introduced method was achieved by the hydrostatic step-pressure micropipette manipulation technique. Additionally the result was reinforced in standard microfluidic shear stress channels. Nevertheless, automated micropipette gave higher sensitivity and less side-effect than the shear stress channel. Using our technique, the probed single cells can be easily picked up and further investigated by other techniques; a definite advantage of the computer controlled micropipette. Our experiments revealed the existence of a sub-population of strongly fibrinogen adherent cells appearing in macrophages and highly represented in dendritic cells, but not observed in monocytes. PMID:25343359

CAVITATION SOUNDS DURING CERVICOTHORACIC SPINAL MANIPULATION

PubMed Central

Mourad, Firas; Zingoni, Andrea; Iorio, Raffaele; Perreault, Thomas; Zacharko, Noah; de las Peñas, César Fernández; Butts, Raymond; Cleland, Joshua A.

2017-01-01

Background No study has previously investigated the side, duration or number of audible cavitation sounds during high-velocity low-amplitude (HVLA) thrust manipulation to the cervicothoracic spine. Purpose The primary purpose was to determine which side of the spine cavitates during cervicothoracic junction (CTJ) HVLA thrust manipulation. Secondary aims were to calculate the average number of cavitations, the duration of cervicothoracic thrust manipulation, and the duration of a single cavitation. Study Design Quasi-experimental study Methods Thirty-two patients with upper trapezius myalgia received two cervicothoracic HVLA thrust manipulations targeting the right and left T1-2 articulation, respectively. Two high sampling rate accelerometers were secured bilaterally 25 mm lateral to midline of the T1-2 interspace. For each manipulation, two audio signals were extracted using Short-Time Fourier Transformation (STFT) and singularly processed via spectrogram calculation in order to evaluate the frequency content and number of instantaneous energy bursts of both signals over time for each side of the CTJ. Result Unilateral cavitation sounds were detected in 53 (91.4%) of 58 cervicothoracic HVLA thrust manipulations and bilateral cavitation sounds were detected in just five (8.6%) of the 58 thrust manipulations; that is, cavitation was significantly (p<0.001) more likely to occur unilaterally than bilaterally. In addition, cavitation was significantly (p<0.0001) more likely to occur on the side contralateral to the clinician's short-lever applicator. The mean number of audible cavitations per manipulation was 4.35 (95% CI 2.88, 5.76). The mean duration of a single manipulation was 60.77 ms (95% CI 28.25, 97.42) and the mean duration of a single audible cavitation was 4.13 ms (95% CI 0.82, 7.46). In addition to single-peak and multi-peak energy bursts, spectrogram analysis also demonstrated high frequency sounds, low frequency sounds, and sounds of multiple frequencies for all 58 manipulations. Discussion Cavitation was significantly more likely to occur unilaterally, and on the side contralateral to the short-lever applicator contact, during cervicothoracic HVLA thrust manipulation. Clinicians should expect multiple cavitation sounds when performing HVLA thrust manipulation to the CTJ. Due to the presence of multi-peak energy bursts and sounds of multiple frequencies, the cavitation hypothesis (i.e. intra-articular gas bubble collapse) alone appears unable to explain all of the audible sounds during HVLA thrust manipulation, and the possibility remains that several phenomena may be occurring simultaneously. Level of Evidence 2b PMID:28900571

Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

PubMed

Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

2013-10-01

Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

Community resources and technologies developed through the NIH Roadmap Epigenomics Program.

PubMed

Satterlee, John S; Beckel-Mitchener, Andrea; McAllister, Kim; Procaccini, Dena C; Rutter, Joni L; Tyson, Frederick L; Chadwick, Lisa Helbling

2015-01-01

This chapter describes resources and technologies generated by the NIH Roadmap Epigenomics Program that may be useful to epigenomics researchers investigating a variety of diseases including cancer. Highlights include reference epigenome maps for a wide variety of human cells and tissues, the development of new technologies for epigenetic assays and imaging, the identification of novel epigenetic modifications, and an improved understanding of the role of epigenetic processes in a diversity of human diseases. We also discuss future needs in this area including exploration of epigenomic variation between individuals, single-cell epigenomics, environmental epigenomics, exploration of the use of surrogate tissues, and improved technologies for epigenome manipulation.

Cell damage evaluation of mammalian cells in cell manipulation by amplified femtosecond ytterbium laser

NASA Astrophysics Data System (ADS)

Hong, Z.-Y.; Iino, T.; Hagihara, H.; Maeno, T.; Okano, K.; Yasukuni, R.; Hosokawa, Y.

2018-03-01

A micrometer-scale explosion with cavitation bubble generation is induced by focusing a femtosecond laser in an aqueous solution. We have proposed to apply the explosion as an impulsive force to manipulate mammalian cells especially in microfluidic chip. Herein, we employed an amplified femtosecond ytterbium laser as an excitation source for the explosion and evaluated cell damage in the manipulation process to clarify the application potential. The damage of C2C12 myoblast cell prepared as a representative mammalian cell was investigated as a function of distance between cell and laser focal point. Although the cell received strong damage on the direct laser irradiation condition, the damage sharply decreased with increasing distance. Since the threshold distance, above which the cell had no damage, was consistent with radius of the cavitation bubble, impact of the cavitation bubble would be a critical factor for the cell damage. The damage had strong nonlinearity in the pulse energy dependence. On the other hand, cell position shift by the impact of the cavitation bubble was almost proportional to the pulse energy. In balance between the cell viability and the cell position shift, we elucidated controllability of the cell manipulation in microfluidic chip.

Laser nanosurgery and manipulation in living cells

NASA Astrophysics Data System (ADS)

Sacconi, Leonardo; Tolic-Norrelykke, Iva M.; Antolini, Renzo; Pavone, Francesco S.

2005-03-01

We present a combination of nonlinear microscopy, laser nanosurgery and optical trapping applied to the 3D imaging and manipulation of intracellular structures in live cells. We use Titanium-sapphire laser pulses for a combined nonlinear microscopy and nanosurgery on microtubules tagged with green fluorescent protein (GFP) in fission yeast. The same laser source is also used to trap small round lipid droplets naturally present in the cell. The trapped droplets are used as handles to exert a pushing force on the nucleus, allowing for a displacement of the nucleus away from its normal position in the center of the cell. We show that nonlinear nanosurgery and optical manipulation can be performed with sub-micrometer precision and without visible collateral damage to the cell. We present this combination as an important tool in cell biology for the manipulation of specific structures in alternative to genetic methods or chemical agents. This technique can be applied to several fundamental problems in cell biology, including the study of dynamics processes in cell division.

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

PubMed

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.

The microcomputer scientific software series 1: the numerical information manipulation system.

Treesearch

Harold M. Rauscher

1983-01-01

The Numerical Information Manipulation System extends the versatility provided by word processing systems for textual data manipulation to mathematical or statistical data in numeric matrix form. Numeric data, stored and processed in the matrix form, may be manipulated in a wide variety of ways. The system allows operations on single elements, entire rows, or columns...

Realization of compact tractor beams using acoustic delay-lines

NASA Astrophysics Data System (ADS)

Marzo, A.; Ghobrial, A.; Cox, L.; Caleap, M.; Croxford, A.; Drinkwater, B. W.

2017-01-01

A method for generating stable ultrasonic levitation of physical matter in air using single beams (also known as tractor beams) is demonstrated. The method encodes the required phase modulation in passive unit cells into which the ultrasonic sources are mounted. These unit cells use waveguides such as straight and coiled tubes to act as delay-lines. It is shown that a static tractor beam can be generated using a single electrical driving signal, and a tractor beam with one-dimensional movement along the propagation direction can be created with two signals. Acoustic tractor beams capable of holding millimeter-sized polymer particles of density 1.25 g/cm3 and fruit-flies (Drosophila) are demonstrated. Based on these design concepts, we show that portable tractor beams can be constructed with simple components that are readily available and easily assembled, enabling applications in industrial contactless manipulation and biophysics.

Topological quantum computing with a very noisy network and local error rates approaching one percent.

PubMed

Nickerson, Naomi H; Li, Ying; Benjamin, Simon C

2013-01-01

A scalable quantum computer could be built by networking together many simple processor cells, thus avoiding the need to create a single complex structure. The difficulty is that realistic quantum links are very error prone. A solution is for cells to repeatedly communicate with each other and so purify any imperfections; however prior studies suggest that the cells themselves must then have prohibitively low internal error rates. Here we describe a method by which even error-prone cells can perform purification: groups of cells generate shared resource states, which then enable stabilization of topologically encoded data. Given a realistically noisy network (≥10% error rate) we find that our protocol can succeed provided that intra-cell error rates for initialisation, state manipulation and measurement are below 0.82%. This level of fidelity is already achievable in several laboratory systems.

A sensible technique to detect mollicutes impurities in human cells cultured in GMP condition.

PubMed

Ugolotti, Elisabetta; Vanni, Irene

2014-01-01

In therapeutic trials the use of manipulated cell cultures for clinical applications is often required. Mollicutes microorganism contamination of tissue cultures is a major problem because it can determine various and severe alterations in cellular function. Thus methods able to detect and trace cell cultures with Mollicutes contamination are needed in the monitoring of cells grown under good manufacturing practice conditions, and cell lines in continuous culture must be tested at regular intervals. We here describe a multiplex quantitative polymerase chain reaction assay able to detect contaminant Mollicutes species in a single-tube reaction through analysis of 16S-23S rRNA intergenic spacer regions and Tuf and P1 cytoadhesin genes. The method shows a sensitivity, specificity, and robustness comparable with the culture and the indicator cell culture as required by the European Pharmacopoeia guidelines and was validated following International Conference on Harmonization guidelines and Food and Drug Administration requirements.

Cryopreservation of human insulin expressing cells macro-encapsulated in a durable therapeutic immunoisolating device theracyte.

PubMed

Yakhnenko, Ilya; Wong, Wallace K; Katkov, Igor I; Itkin-Ansari, Pamela

2012-01-01

Encapsulating insulin producing cells (INPCs) in an immunoisolation device have been shown to cure diabetes in rodents without the need for immunosuppression. However, micro-encapsulation in semi-solid gels raises longevity and safety concerns for future use of stem cell derived INPCs. We have focused on a durable and retrievable macro-encapsulation (> 10(6) cells) device (TheraCyte). Cryopreservation (CP) of cells preloaded into the device is highly desirable but may require prolonged exposure to cryoprotectants during loading and post-thaw manipulations. Here, we are reporting survival and function of a human islet cell line frozen as single cells or as islet-like cell clusters. The non-clusterized cells exhibited high cryosurvival after prolonged pre-freeze or post-thaw exposure to 10 percent DMSO. However, both clusterization and especially loading INPCs into the device reduced viable yield even without CP. The survived cryopreserved macro-encapsulated INPCs remained fully functional suggesting that CP of macro-encapsulated cells is a promising tool for cell based therapies.

Analysis of a minimal Rho-GTPase circuit regulating cell shape

NASA Astrophysics Data System (ADS)

Holmes, William R.; Edelstein-Keshet, Leah

2016-08-01

Networks of Rho-family GTPases regulate eukaryotic cell polarization and motility by controlling assembly and contraction of the cytoskeleton. The mutually inhibitory Rac-Rho circuit is emerging as a central, regulatory hub that can affect the shape and motility phenotype of eukaryotic cells. Recent experimental manipulation of the amounts of Rac and Rho or their regulators (guanine nucleotide-exchange factors, GTPase-activating proteins, guanine nucleotide dissociation inhibitors) have been shown to bias the prevalence of these different states and promote transitions between them. Here we show that part of this data can be understood in terms of inherent Rac-Rho mutually inhibitory dynamics. We analyze a spatio-temporal mathematical model of Rac-Rho dynamics to produce a detailed set of predictions of how parameters such as GTPase rates of activation and total amounts affect cell decisions (such as Rho-dominated contraction, Rac-dominated spreading, and spatially segregated Rac-Rho polarization). We find that in some parameter regimes, a cell can take on any of these three fates depending on its environment or stimuli. We also predict how experimental manipulations (corresponding to parameter variations) can affect cell shapes observed. Our methods are based on local perturbation analysis (a kind of nonlinear stability analysis), and an approximation of nonlinear feedback by sharp switches. We compare the Rac-Rho model to an even simpler single-GTPase (‘wave-pinning’) model and demonstrate that the overall behavior is inherent to GTPase properties, rather than stemming solely from network topology.

512-Channel and 13-Region Simultaneous Recordings Coupled with Optogenetic Manipulation in Freely Behaving Mice

PubMed Central

Xie, Kun; Fox, Grace E.; Liu, Jun; Tsien, Joe Z.

2016-01-01

The development of technologies capable of recording both single-unit activity and local field potentials (LFPs) over a wide range of brain circuits in freely behaving animals is the key to constructing brain activity maps. Although mice are the most popular mammalian genetic model, in vivo neural recording has been traditionally limited to smaller channel count and fewer brain structures because of the mouse’s small size and thin skull. Here, we describe a 512-channel tetrode system that allows us to record simultaneously over a dozen cortical and subcortical structures in behaving mice. This new technique offers two major advantages – namely, the ultra-low cost and the do-it-yourself flexibility for targeting any combination of many brain areas. We show the successful recordings of both single units and LFPs from 13 distinct neural circuits of the mouse brain, including subregions of the anterior cingulate cortices, retrosplenial cortices, somatosensory cortices, secondary auditory cortex, hippocampal CA1, dentate gyrus, subiculum, lateral entorhinal cortex, perirhinal cortex, and prelimbic cortex. This 512-channel system can also be combined with Cre-lox neurogenetics and optogenetics to further examine interactions between genes, cell types, and circuit dynamics across a wide range of brain structures. Finally, we demonstrate that complex stimuli – such as an earthquake and fear-inducing foot-shock – trigger firing changes in all of the 13 brain regions recorded, supporting the notion that neural code is highly distributed. In addition, we show that localized optogenetic manipulation in any given brain region could disrupt network oscillations and caused changes in single-unit firing patterns in a brain-wide manner, thereby raising the cautionary note of the interpretation of optogenetically manipulated behaviors. PMID:27378865

Investigating cell-substrate and cell-cell interactions by means of single-cell-probe force spectroscopy.

PubMed

Moreno-Cencerrado, Alberto; Iturri, Jagoba; Pecorari, Ilaria; D M Vivanco, Maria; Sbaizero, Orfeo; Toca-Herrera, José L

2017-01-01

Cell adhesion forces are typically a mixture of specific and nonspecific cell-substrate and cell-cell interactions. In order to resolve these phenomena, Atomic Force Microscopy appears as a powerful device which can measure cell parameters by means of manipulation of single cells. This method, commonly known as cell-probe force spectroscopy, allows us to control the force applied, the area of interest, the approach/retracting speed, the force rate, and the time of interaction. Here, we developed a novel approach for in situ cantilever cell capturing and measurement of specific cell interactions. In particular, we present a new setup consisting of two different half-surfaces coated either with recrystallized SbpA bacterial cell surface layer proteins (S-layers) or integrin binding Fibronectin, on which MCF-7 breast cancer cells are incubated. The presence of a clear physical boundary between both surfaces benefits for a quick detection of the region under analysis. Thus, quantitative results about SbpA-cell and Fibronectin-cell adhesion forces as a function of the contact time are described. Additionally, the importance of the cell spreading in cell-cell interactions has been studied for surfaces coated with two different Fibronectin concentrations: 20 μg/mL (FN20) and 100 μg/mL (FN100), which impact the number of substrate receptors. Microsc. Res. Tech. 80:124-130, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Holographic imaging and photostimulation of neural activity.

PubMed

Yang, Weijian; Yuste, Rafael

2018-06-01

Optical imaging methods are powerful tools in neuroscience as they can systematically monitor the activity of neuronal populations with high spatiotemporal resolution using calcium or voltage indicators. Moreover, caged compounds and optogenetic actuators enable to optically manipulate neural activity. Among optical methods, computer-generated holography offers an enormous flexibility to sculpt the excitation light in three-dimensions (3D), particularly when combined with two-photon light sources. By projecting holographic light patterns on the sample, the activity of multiple neurons across a 3D brain volume can be simultaneously imaged or optically manipulated with single-cell precision. This flexibility makes two-photon holographic microscopy an ideal all-optical platform to simultaneously read and write activity in neuronal populations in vivo in 3D, a critical ability to dissect the function of neural circuits. Copyright © 2018 Elsevier Ltd. All rights reserved.

A review on optical actuators for microfluidic systems

NASA Astrophysics Data System (ADS)

Yang, Tie; Chen, Yue; Minzioni, Paolo

2017-12-01

During the last few decades microfluidic systems have become more and more popular and their relevance in different fields is continually growing. In fact, the use of microchannels allows a significant reduction of the required sample-volume and opens the way to a completely new set of possible investigations, including the study of the properties of cells, the development of new cells’ separation techniques and the analysis of single-cell proteins. One of the main differences between microscopic and macroscopic systems is obviously dictated by the need for suitable actuation mechanisms, which should allow precise control of microscopic fluid volumes and of micro-samples inside the fluid. Even if both syringe-pump and pneumatic-pump technologies significantly evolved and they currently enable sub-μL samples control, completely new approaches were recently developed for the manipulation of samples inside the microchannel. This review is dedicated to describing different kinds of optical actuators that can be applied in microfluidic systems for sample manipulation as well as for pumping. The basic principles underlying the optical actuation mechanisms will be described first, and then several experimental demonstrations will be reviewed and compared.

Optical Manipulation of Symbiotic Chlorella in Paramecium Bursaria Using a Fiber Axicon Microlens

NASA Astrophysics Data System (ADS)

Taguchi, K.; Hirota, S.; Nakayama, H.; Kunugihara, D.; Mihara, Y.

2012-03-01

In this paper, chemically etched axicon fiber was proposed for laser trapping of symbiotic chlorella from paramecium bursaria. We fabricated axicon micro lenses on a single-mode bare optical fiber by selective chemical etching technique. The laser beam from fiber axicon microlens was strongly focused and optical forces were sufficient to move a symbiotic chlorella. From experimental results, it was found that our proposed fiber axicon microlens was a promising tool for cell trapping without physical contact.

DNA Damage and Oxidative Stress in Dyskeratosis Congenita: Analysis of Pathways and Therapeutic Stategies Using CPISPR and iPSC Model Systems

DTIC Science & Technology

2017-06-01

Milestone Achieved: HRPO/ACURO Approval 6 Finished Major Task 2 CRISPR knockout/RNAseq Viral infection/prep 3-6 CRISPR KO virus library prep...finished; RNA-Seq: ~75% Cell manipulation 3-6 CRISPR KO virus infection: 50%; Single cDNA infections: finished Bioinformatics 1 CRISPR KO library...characterization 1-3 Finished Update: production of iPSC clones harboring DC mutations generated by CRISPR : Design 1 Finished Update: production of

Precise Spatiotemporal Control of Optogenetic Activation Using an Acousto-Optic Device

PubMed Central

Guo, Yanmeng; Song, Peipei; Zhang, Xiaohui; Zeng, Shaoqun; Wang, Zuoren

2011-01-01

Light activation and inactivation of neurons by optogenetic techniques has emerged as an important tool for studying neural circuit function. To achieve a high resolution, new methods are being developed to selectively manipulate the activity of individual neurons. Here, we report that the combination of an acousto-optic device (AOD) and single-photon laser was used to achieve rapid and precise spatiotemporal control of light stimulation at multiple points in a neural circuit with millisecond time resolution. The performance of this system in activating ChIEF expressed on HEK 293 cells as well as cultured neurons was first evaluated, and the laser stimulation patterns were optimized. Next, the spatiotemporally selective manipulation of multiple neurons was achieved in a precise manner. Finally, we demonstrated the versatility of this high-resolution method in dissecting neural circuits both in the mouse cortical slice and the Drosophila brain in vivo. Taken together, our results show that the combination of AOD-assisted laser stimulation and optogenetic tools provides a flexible solution for manipulating neuronal activity at high efficiency and with high temporal precision. PMID:22174813

Electric-Field Control of Oxygen Vacancies and Magnetic Phase Transition in a Cobaltite/Manganite Bilayer

NASA Astrophysics Data System (ADS)

Cui, B.; Song, C.; Li, F.; Zhong, X. Y.; Wang, Z. C.; Werner, P.; Gu, Y. D.; Wu, H. Q.; Saleem, M. S.; Parkin, S. S. P.; Pan, F.

2017-10-01

Manipulation of oxygen vacancies (VO ) in single oxide layers by varying the electric field can result in significant modulation of the ground state. However, in many oxide multilayers with strong application potentials, e.g., ferroelectric tunnel junctions and solid-oxide fuel cells, understanding VO behavior in various layers under an applied electric field remains a challenge, owing to complex VO transport between different layers. By sweeping the external voltage, a reversible manipulation of VO and a corresponding fixed magnetic phase transition sequence in cobaltite/manganite (SrCoO3 -x/La0.45Sr0.55MnO3 -y ) heterostructures are reported. The magnetic phase transition sequence confirms that the priority of electric-field-induced VO formation or annihilation in the complex bilayer system is mainly determined by the VO formation energies and Gibbs free-energy differences, which is supported by theoretical analysis. We not only realize a reversible manipulation of the magnetic phase transition in an oxide bilayer but also provide insight into the electric-field control of VO engineering in heterostructures.

Hydrodynamics in Cell Studies

PubMed Central

2018-01-01

Hydrodynamic phenomena are ubiquitous in living organisms and can be used to manipulate cells or emulate physiological microenvironments experienced in vivo. Hydrodynamic effects influence multiple cellular properties and processes, including cell morphology, intracellular processes, cell–cell signaling cascades and reaction kinetics, and play an important role at the single-cell, multicellular, and organ level. Selected hydrodynamic effects can also be leveraged to control mechanical stresses, analyte transport, as well as local temperature within cellular microenvironments. With a better understanding of fluid mechanics at the micrometer-length scale and the advent of microfluidic technologies, a new generation of experimental tools that provide control over cellular microenvironments and emulate physiological conditions with exquisite accuracy is now emerging. Accordingly, we believe that it is timely to assess the concepts underlying hydrodynamic control of cellular microenvironments and their applications and provide some perspective on the future of such tools in in vitro cell-culture models. Generally, we describe the interplay between living cells, hydrodynamic stressors, and fluid flow-induced effects imposed on the cells. This interplay results in a broad range of chemical, biological, and physical phenomena in and around cells. More specifically, we describe and formulate the underlying physics of hydrodynamic phenomena affecting both adhered and suspended cells. Moreover, we provide an overview of representative studies that leverage hydrodynamic effects in the context of single-cell studies within microfluidic systems. PMID:29420889

Ultrashort laser pulse cell manipulation using nano- and micro- materials

NASA Astrophysics Data System (ADS)

Schomaker, Markus; Killian, Doreen; Willenbrock, Saskia; Diebold, Eric; Mazur, Eric; Bintig, Willem; Ngezahayo, Anaclet; Nolte, Ingo; Murua Escobar, Hugo; Junghanß, Christian; Lubatschowski, Holger; Heisterkamp, Alexander

2010-08-01

The delivery of extra cellular molecules into cells is essential for cell manipulation. For this purpose genetic materials (DNA/RNA) or proteins have to overcome the impermeable cell membrane. To increase the delivery efficiency and cell viability of common methods different nano- and micro material based approaches were applied. To manipulate the cells, the membrane is in contact with the biocompatible material. Due to a field enhancement of the laser light at the material and the resulting effect the cell membrane gets perforated and extracellular molecules can diffuse into the cytoplasm. Membrane impermeable dyes, fluorescent labelled siRNA, as well as plasmid vectors encoded for GFP expression were used as an indicator for successful perforation or transfection, respectively. Dependent on the used material, perforation efficiencies over 90 % with a cell viability of about 80 % can be achieved. Additionally, we observed similar efficiencies for siRNA transfection. Due to the larger molecule size and the essential transport of the DNA into the nucleus cells are more difficult to transfect with GFP plasmid vectors. Proof of principle experiments show promising and adequate efficiencies by applying micro materials for plasmid vector transfection. For all methods a weakly focused fs laser beam is used to enable a high manipulation throughput for adherent and suspension cells. Furthermore, with these alternative optical manipulation methods it is possible to perforate the membrane of sensitive cell types such as primary and stem cells with a high viability.

Flow-assisted single-beam optothermal manipulation of microparticles.

PubMed

Liu, Yangyang; Poon, Andrew W

2010-08-16

An optothermal tweezer was developed with a single-beam laser at 1550 nm for manipulation of colloidal microparticles. Strong absorption in water can thermally induce a localized flow, which exerts a Stokes' drag on the particles that complements the gradient force. Long-range capturing of 6 microm polystyrene particles over approximately 176 microm was observed with a tweezing power of approximately 7 mW. Transportation and levitation, targeted deposition and selective levitation of particles were explored to experimentally demonstrate the versatility of the optothermal tweezer as a multipurpose particle manipulation tool.

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers.

PubMed

Roche, Stuart M; Gumucio, Jonathan P; Brooks, Susan V; Mendias, Christopher L; Claflin, Dennis R

2015-06-16

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gross, Benjamin J.; El-Naggar, Mohamed Y., E-mail: mnaggar@usc.edu; Molecular and Computational Biology Section, Department of Biological Sciences, University of Southern California, Los Angeles, California 90089-0484

2015-06-15

Metal-reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. This process is one of the earliest forms of respiration on earth and has significant environmental and technological implications. By performing electron transfer to electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. Understanding such a complex biotic-abiotic interaction necessitates the development of tools capable of probing extracellular electron transfer down to the level of single cells. Here, we describe anmore » experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect the respiration current correlated with cell-electrode contact. We demonstrate the system with single cell measurements of the dissimilatory-metal reducing bacterium Shewanella oneidensis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer-membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. In addition to the application for extracellular electron transfer studies, the ability to electronically measure cell-specific respiration rates may provide answers for a variety of fundamental microbial physiology questions.« less

Project Plan 7930 Cell G PaR Remote Handling System Replacement

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kinney, Kathryn A

2009-10-01

For over 40 years the US Department of Energy (DOE) and its predecessors have made Californium-252 ({sup 252}Cf) available for a wide range of industries including medical, nuclear fuels, mining, military and national security. The Radiochemical Engineering Development Center (REDC) located within the Oak Ridge National Laboratory (ORNL) processes irradiated production targets from the High Flux Isotope Reactor (HFIR). Operations in Building 7930, Cell G provide over 70% of the world's demand for {sup 252}Cf. Building 7930 was constructed and equipped in the mid-1960s. Current operations for {sup 252}Cf processing in Building 7930, Cell G require use of through-the-wall manipulatorsmore » and the PaR Remote Handling System. Maintenance and repairs for the manipulators is readily accomplished by removal of the manipulator and relocation to a repair shop where hands-on work can be performed in glove boxes. Contamination inside cell G does not currently allow manned entry and no provisions were created for a maintenance area inside the cell. There has been no maintenance of the PaR system or upgrades, leaving operations vulnerable should the system have a catastrophic failure. The Cell G PaR system is currently being operated in a run to failure mode. As the manipulator is now 40+ years old there is significant risk in this method of operation. In 2006 an assessment was completed that resulted in recommendations for replacing the manipulator operator control and power centers which are used to control and power the PaR manipulator in Cell G. In mid-2008 the chain for the bridge drive failed and subsequent examinations indicated several damaged links (see Figure 1). To continue operations the PaR manipulator arm is being used to push and pull the bridge as a workaround. A retrieval tool was fabricated, tested and staged inside Cell G that will allow positioning of the bridge and manipulator arm for removal from the cell should the PaR system completely fail. A fully functioning and reliable Par manipulator arm is necessary for uninterrupted {sup 252}Cf operations; a fully-functioning bridge is needed for the system to function as intended.« less

Development of a Single-Cell Migration and Extravasation Platform through Selective Surface Modification.

PubMed

Roberts, Steven A; Waziri, Allen E; Agrawal, Nitin

2016-03-01

Cell migration through three-dimensional (3D) tissue spaces is integral to many biological and pathological processes, including metastasis. Circulating tumor cells (CTCs) are phenotypically heterogeneous, and in vitro analysis of their extravasation behavior is often impeded by the inability to establish complex tissue-like extracellular matrix (ECM) environments and chemotactic gradients within microfluidic devices. We have developed a novel microfluidic strategy to manipulate surface properties of enclosed microchannels and create 3D ECM structures for real-time observation of individual migrating cells. The wettability of selective interconnected channels is controlled by a plasma pulse, enabling the incorporation of ECM exclusively within the transmigration regions. We applied this approach to collectively analyze CTC-endothelial adhesion, trans-endothelial migration, and subsequent motility of breast cancer cells (MDA-MB-231) through a 3D ECM under artificial gradients of SDF-1α. We observed migration velocities ranging from 5.12 to 12.8 μm/h, which closely correspond to single-cell migration in collagen blocks, but are significantly faster than the migration of cell aggregates. The compartmentalized microchannels separated by the porous ECM makes this in vitro assay versatile and suitable for a variety of applications such as inflammation studies, drug screening, and coculture interactions.

Protein kinase A inhibitor, H89, significantly enhances survival rate of dissociated human embryonic stem cells following cryopreservation.

PubMed

Zhang, Liang; Xu, Yanqing; Xu, Jiandong; Wei, Yuping; Xu, Xia

2016-10-01

Human embryonic stem cells (hESCs) have huge potential for establishment of disease models and for treating degenerative diseases. However, the extremely low survival level of dissociated hESCs following cryopreservation is been a tremendous problem to allow for their rapid expansion, genetic manipulation and future medical applications. In this study, we have aimed to develop an efficient strategy to improve survival of dissociated hESCs after cryopreservation. Human embryonic stem cells (H9 line), dissociated into single cells, were cryopreserved using the slow-freezing method. Viable cells and their colony numbers in culture after cryopreservation were evaluated when treated with protein kinase A inhibitor H89. Western blotting was carried out to investigate mechanisms of low survival levels of dissociated hESCs following cryopreservation. Immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR), in vitro and in vivo differentiation were performed to testify to pluripotency and differentiation ability of hte cryopreserved cells treated with H89. H89 significantly improved survival level of dissociated hESCs after cryopreservation through ROCK inhibition. H89-treated cells still maintained their pluripotency and differentiation capacity. This new approach for cryopreservation of single hESCs, using H89, can promote potential use of hESCs in regenerative medicine in the future. © 2016 John Wiley & Sons Ltd.

The effect of talocrural joint manipulation on range of motion at the ankle.

PubMed

Fryer, Gary A; Mudge, Jacob M; McLaughlin, Patrick A

2002-01-01

To determine whether a single high-velocity, low-amplitude thrust manipulation to the talocrural joint altered ankle range of motion. A randomized, controlled and blinded study. Asymptomatic male and female volunteers (N = 41). Subjects were randomly assigned into either an experimental group (n = 20) or a control group (n = 21). Both ankles of subjects in the experimental group were manipulated by using a single high-velocity, low-amplitude thrust to the talocrural joint. Pretest and posttest measurements of passive dorsiflexion range of motion were taken. No significant changes in dorsiflexion range of motion were detected between manipulated ankles and those of control subjects. A significantly greater pretest dorsiflexion range of motion existed in those ankles in which manipulation produced an audible cavitation. Manipulation of the ankle does not increase dorsiflexion range of motion in asymptomatic subjects. Ankles that displayed a greater pretest range of dorsiflexion were more likely to cavitate, raising the possibility that ligament laxity may be associated with the tendency for ankles to cavitate.

Kinematics Modelling of Tendon-Driven Continuum Manipulator with Crossed Notches

NASA Astrophysics Data System (ADS)

Yang, Z. X.; Yang, W. L.; Du, Z. J.

2018-03-01

Single port surgical robot (SPSR) is a giant leap in the development of minimally invasive surgical robot. An innovative manipulator with high control accuracy and good kinematic dexterity can reduce wound, expedite recovery, and improve the success rate. This paper presents a tendon-driven continuum manipulator with crossed notches. This manipulator has two degrees of freedom (DOF), which possesses good flexibility and high capacity. Then based on cantilever beam theory, a mechanics model is proposed, which connects external force and deformation of a single flexible ring (SFR). By calculating the deformation of each SFR, the manipulator is considered as a series robot whose joint numbers is equal to SFR numbers, and the kinematics model is established through Denavit-Hartenberg (D-H) procedure. In this paper, the total manipulator is described as a curve tube whose curvature is increased from tip to base. Experiments were conducted and the comparison between theoretical and actual results proved the rationality of the models.

Towards circuit optogenetics.

PubMed

Chen, I-Wen; Papagiakoumou, Eirini; Emiliani, Valentina

2018-06-01

Optogenetics neuronal targeting combined with single-photon wide-field illumination has already proved its enormous potential in neuroscience, enabling the optical control of entire neuronal networks and disentangling their role in the control of specific behaviors. However, establishing how a single or a sub-set of neurons controls a specific behavior, or how functionally identical neurons are connected in a particular task, or yet how behaviors can be modified in real-time by the complex wiring diagram of neuronal connections requires more sophisticated approaches enabling to drive neuronal circuits activity with single-cell precision and millisecond temporal resolution. This has motivated on one side the development of flexible optical methods for two-photon (2P) optogenetic activation using either, or a hybrid of two approaches: scanning and parallel illumination. On the other side, it has stimulated the engineering of new opsins with modified spectral characteristics, channel kinetics and spatial distribution of expression, offering the necessary flexibility of choosing the appropriate opsin for each application. The need for optical manipulation of multiple targets with millisecond temporal resolution has imposed three-dimension (3D) parallel holographic illumination as the technique of choice for optical control of neuronal circuits organized in 3D. Today 3D parallel illumination exists in several complementary variants, each with a different degree of simplicity, light uniformity, temporal precision and axial resolution. In parallel, the possibility to reach hundreds of targets in 3D volumes has prompted the development of low-repetition rate amplified laser sources enabling high peak power, while keeping low average power for stimulating each cell. All together those progresses open the way for a precise optical manipulation of neuronal circuits with unprecedented precision and flexibility. Copyright © 2018 Elsevier Ltd. All rights reserved.

A planarian p53 homolog regulates proliferation and self-renewal in adult stem cell lineages.

PubMed

Pearson, Bret J; Sánchez Alvarado, Alejandro

2010-01-01

The functions of adult stem cells and tumor suppressor genes are known to intersect. However, when and how tumor suppressors function in the lineages produced by adult stem cells is unknown. With a large population of stem cells that can be manipulated and studied in vivo, the freshwater planarian is an ideal system with which to investigate these questions. Here, we focus on the tumor suppressor p53, homologs of which have no known role in stem cell biology in any invertebrate examined thus far. Planaria have a single p53 family member, Smed-p53, which is predominantly expressed in newly made stem cell progeny. When Smed-p53 is targeted by RNAi, the stem cell population increases at the expense of progeny, resulting in hyper-proliferation. However, ultimately the stem cell population fails to self-renew. Our results suggest that prior to the vertebrates, an ancestral p53-like molecule already had functions in stem cell proliferation control and self-renewal.

Substrate stiffness regulates cadherin-dependent collective migration through myosin-II contractility

PubMed Central

Ng, Mei Rosa; Besser, Achim

2012-01-01

The mechanical microenvironment is known to influence single-cell migration; however, the extent to which mechanical cues affect collective migration of adherent cells is not well understood. We measured the effects of varying substrate compliance on individual cell migratory properties in an epithelial wound-healing assay. Increasing substrate stiffness increased collective cell migration speed, persistence, and directionality as well as the coordination of cell movements. Dynamic analysis revealed that wounding initiated a wave of motion coordination from the wound edge into the sheet. This was accompanied by a front-to-back gradient of myosin-II activation and establishment of cell polarity. The propagation was faster and farther reaching on stiff substrates, indicating that substrate stiffness affects the transmission of directional cues. Manipulation of myosin-II activity and cadherin–catenin complexes revealed that this transmission is mediated by coupling of contractile forces between neighboring cells. Thus, our findings suggest that the mechanical environment integrates in a feedback with cell contractility and cell–cell adhesion to regulate collective migration. PMID:23091067

Clinical-scale selection and viral transduction of human naïve and central memory CD8+ T cells for adoptive cell therapy of cancer patients.

PubMed

Casati, Anna; Varghaei-Nahvi, Azam; Feldman, Steven Alexander; Assenmacher, Mario; Rosenberg, Steven Aaron; Dudley, Mark Edward; Scheffold, Alexander

2013-10-01

The adoptive transfer of lymphocytes genetically engineered to express tumor-specific antigen receptors is a potent strategy to treat cancer patients. T lymphocyte subsets, such as naïve or central memory T cells, selected in vitro prior to genetic engineering have been extensively investigated in preclinical mouse models, where they demonstrated improved therapeutic efficacy. However, so far, this is challenging to realize in the clinical setting, since good manufacturing practices (GMP) procedures for complex cell sorting and genetic manipulation are limited. To be able to directly compare the immunological attributes and therapeutic efficacy of naïve (T(N)) and central memory (T(CM)) CD8(+) T cells, we investigated clinical-scale procedures for their parallel selection and in vitro manipulation. We also evaluated currently available GMP-grade reagents for stimulation of T cell subsets, including a new type of anti-CD3/anti-CD28 nanomatrix. An optimized protocol was established for the isolation of both CD8(+) T(N) cells (CD4(-)CD62L(+)CD45RA(+)) and CD8(+) T(CM) (CD4(-)CD62L(+)CD45RA(-)) from a single patient. The highly enriched T cell subsets can be efficiently transduced and expanded to large cell numbers, sufficient for clinical applications and equivalent to or better than current cell and gene therapy approaches with unselected lymphocyte populations. The GMP protocols for selection of T(N) and T(CM) we reported here will be the basis for clinical trials analyzing safety, in vivo persistence and clinical efficacy in cancer patients and will help to generate a more reliable and efficacious cellular product.

Droplet Array-Based 3D Coculture System for High-Throughput Tumor Angiogenesis Assay.

PubMed

Du, Xiaohui; Li, Wanming; Du, Guansheng; Cho, Hansang; Yu, Min; Fang, Qun; Lee, Luke P; Fang, Jin

2018-03-06

Angiogenesis is critical for tumor progression and metastasis, and it progresses through orchestral multicellular interactions. Thus, there is urgent demand for high-throughput tumor angiogenesis assays for concurrent examination of multiple factors. For investigating tumor angiogenesis, we developed a microfluidic droplet array-based cell-coculture system comprising a two-layer polydimethylsiloxane chip featuring 6 × 9 paired-well arrays and an automated droplet-manipulation device. In each droplet-pair unit, tumor cells were cultured in 3D in one droplet by mixing cell suspensions with Matrigel, and in the other droplet, human umbilical vein endothelial cells (HUVECs) were cultured in 2D. Droplets were fused by a newly developed fusion method, and tumor angiogenesis was assayed by coculturing tumor cells and HUVECs in the fused droplet units. The 3D-cultured tumor cells formed aggregates harboring a hypoxic center-as observed in vivo-and secreted more vascular endothelial growth factor (VEGF) and more strongly induced HUVEC tubule formation than did 2D-cultured tumor cells. Our single array supported 54 assays in parallel. The angiogenic potentials of distinct tumor cells and their differential responses to antiangiogenesis agent, Fingolimod, could be investigated without mutual interference in a single array. Our droplet-based assay is convenient to evaluate multicellular interaction in high throughput in the context of tumor sprouting angiogenesis, and we envision that the assay can be extensively implementable for studying other cell-cell interactions.

Novel physical chemistry approaches in biophysical researches with advanced application of lasers: Detection and manipulation.

PubMed

Iwata, Koichi; Terazima, Masahide; Masuhara, Hiroshi

2018-02-01

Novel methodologies utilizing pulsed or intense CW irradiation obtained from lasers have a major impact on biological sciences. In this article, recent development in biophysical researches fully utilizing the laser irradiation is described for three topics, time-resolved fluorescence spectroscopy, time-resolved thermodynamics, and manipulation of the biological assemblies by intense laser irradiation. First, experimental techniques for time-resolved fluorescence spectroscopy are concisely explained in Section 2. As an example of the recent application of time-resolved fluorescence spectroscopy to biological systems, evaluation of the viscosity of lipid bilayer membranes is described. The results of the spectroscopic experiments strongly suggest the presence of heterogeneous membrane structure with two different viscosity values in liposomes formed by a single phospholipid. Section 3 covers the time-resolved thermodynamics. Thermodynamical properties are important to characterize biomolecules. However, measurement of these quantities for short-lived intermediate species has been impossible by traditional thermodynamical techniques. Recently, development of a spectroscopic method based on the transient grating method enables us to measure these quantities and also to elucidate reaction kinetics which cannot be detected by other spectroscopic methods. The principle of the measurements and applications to some protein reactions are reviewed. Manipulation and fabrication of supramolecues, amino acids, proteins, and living cells by intense laser irradiation are described in Section 4. Unconventional assembly, crystallization and growth, amyloid fibril formation, and living cell manipulation are achieved by CW laser trapping and femtosecond laser-induced cavitation bubbling. Their spatio-temporal controllability is opening a new avenue in the relevant molecular and bioscience research fields. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato. Copyright © 2017. Published by Elsevier B.V.

Quantitative analysis of circadian single cell oscillations in response to temperature

PubMed Central

Kramer, Achim; Herzel, Hanspeter

2018-01-01

Body temperature rhythms synchronize circadian oscillations in different tissues, depending on the degree of cellular coupling: the responsiveness to temperature is higher when single circadian oscillators are uncoupled. So far, the role of coupling in temperature responsiveness has only been studied in organotypic tissue slices of the central circadian pacemaker, because it has been assumed that peripheral target organs behave like uncoupled multicellular oscillators. Since recent studies indicate that some peripheral tissues may exhibit cellular coupling as well, we asked whether peripheral network dynamics also influence temperature responsiveness. Using a novel technique for long-term, high-resolution bioluminescence imaging of primary cultured cells, exposed to repeated temperature cycles, we were able to quantitatively measure period, phase, and amplitude of central (suprachiasmatic nuclei neuron dispersals) and peripheral (mouse ear fibroblasts) single cell oscillations in response to temperature. Employing temperature cycles of different lengths, and different cell densities, we found that some circadian characteristics appear cell-autonomous, e.g. period responses, while others seem to depend on the quality/degree of cellular communication, e.g. phase relationships, robustness of the oscillation, and amplitude. Overall, our findings indicate a strong dependence on the cell’s ability for intercellular communication, which is not only true for neuronal pacemakers, but, importantly, also for cells in peripheral tissues. Hence, they stress the importance of comparative studies that evaluate the degree of coupling in a given tissue, before it may be used effectively as a target for meaningful circadian manipulation. PMID:29293562

Orienting Paramecium with intense static magnetic fields

NASA Astrophysics Data System (ADS)

Valles, James M., Jr.; Guevorkian, Karine; Quindel, Carl

2004-03-01

Recent experiments on cell division suggest the application of intense static magnetic fields as a novel tool for the manipulation of biological systems [1]. The magnetic field appears to couple to the intrinsic anisotropies in the diamagnetic components of the cells. Here, we present measurements of the intrinsic average diamagnetic anisotropy of the whole single celled ciliate, Paramecium Caudatum. Magnetic fields, 2.5 T < B < 8 T were applied to immobilized (non-swimming) Paramecium Caudatum that were suspended in a density matched medium. The organisms align with their long axis parallel to the applied magnetic field. Their intrinsic diamagnetic anisotropy is 3x10-11 in cgs units. We will discuss the implications of these results for employing magnetic fields to probe the behavior of swimming Paramecium. [1] J. M. Valles, Jr. et al., Expt. Cell Res.274, 112-118 (2002).

Micro-particle manipulation by single beam acoustic tweezers based on hydrothermal PZT thick film.

PubMed

Zhu, Benpeng; Xu, Jiong; Li, Ying; Wang, Tian; Xiong, Ke; Lee, Changyang; Yang, Xiaofei; Shiiba, Michihisa; Takeuchi, Shinichi; Zhou, Qifa; Shung, K Kirk

2016-03-01

Single-beam acoustic tweezers (SBAT), used in laboratory-on-a-chip (LOC) device has promising implications for an individual micro-particle contactless manipulation. In this study, a freestanding hydrothermal PZT thick film with excellent piezoelectric property (d 33 = 270pC/N and k t = 0.51) was employed for SBAT applications and a press-focusing technology was introduced. The obtained SBAT, acting at an operational frequency of 50MHz, a low f-number (∼0.9), demonstrated the capability to trap and manipulate a micro-particle sized 10μm in the distilled water. These results suggest that such a device has great potential as a manipulator for a wide range of biomedical and chemical science applications.

Statistical numeracy as a moderator of (pseudo)contingency effects on decision behavior.

PubMed

Fleig, Hanna; Meiser, Thorsten; Ettlin, Florence; Rummel, Jan

2017-03-01

Pseudocontingencies denote contingency estimates inferred from base rates rather than from cell frequencies. We examined the role of statistical numeracy for effects of such fallible but adaptive inferences on choice behavior. In Experiment 1, we provided information on single observations as well as on base rates and tracked participants' eye movements. In Experiment 2, we manipulated the availability of information on cell frequencies and base rates between conditions. Our results demonstrate that a focus on base rates rather than cell frequencies benefits pseudocontingency effects. Learners who are more proficient in (conditional) probability calculation prefer to rely on cell frequencies in order to judge contingencies, though, as was evident from their gaze behavior. If cell frequencies are available in summarized format, they may infer the true contingency between options and outcomes. Otherwise, however, even highly numerate learners are susceptible to pseudocontingency effects. Copyright © 2017 Elsevier B.V. All rights reserved.

An integrated open-cavity system for magnetic bead manipulation.

PubMed

Abu-Nimeh, F T; Salem, F M

2013-02-01

Superparamagnetic beads are increasingly used in biomedical assays to manipulate, transport, and maneuver biomaterials. We present a low-cost integrated system designed in bulk CMOS to manipulate and separate biomedical magnetic beads. The system consists of 8 × 8 coil-arrays suitable for single bead manipulation, or collaborative multi-bead manipulation, using pseudo-parallel executions. We demonstrate the flexibility of the design in terms of different coil sizes, DC current levels, and layout techniques. In one array module example, the size of a single coil is 30 μm × 30 μm and the full array occupies an area of 248 μm × 248 μm in 0.5 μm CMOS technology. The programmable DC current source supports 8 discrete levels up to 1.5 mA. The total power consumption of the entire module is 9 mW when running at full power.

76 FR 51038 - Draft Guidance for Industry: Cell Selection Devices for Point of Care Production of Minimally...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-17

...; formerly Docket No. 2007D-0290] Draft Guidance for Industry: Cell Selection Devices for Point of Care Production of Minimally Manipulated Autologous Peripheral Blood Stem Cells; Withdrawal of Draft Guidance...: Cell Selection Devices for Point of Care Production of Minimally Manipulated Autologous Peripheral...

A minimally invasive optical trapping system to understand cellular interactions at onset of an immune response

PubMed Central

Glass, David G.; McAlinden, Niall; Millington, Owain R.

2017-01-01

T-cells and antigen presenting cells are an essential part of the adaptive immune response system and how they interact is crucial in how the body effectively fights infection or responds to vaccines. Much of the experimental work studying interaction forces between cells has looked at the average properties of bulk samples of cells or applied microscopy to image the dynamic contact between these cells. In this paper we present a novel optical trapping technique for interrogating the force of this interaction and measuring relative interaction forces at the single-cell level. A triple-spot optical trap is used to directly manipulate the cells of interest without introducing foreign bodies such as beads to the system. The optical trap is used to directly control the initiation of cell-cell contact and, subsequently to terminate the interaction at a defined time point. The laser beam power required to separate immune cell pairs is determined and correlates with the force applied by the optical trap. As proof of concept, the antigen-specific increase in interaction force between a dendritic cell and a specific T-cell is demonstrated. Furthermore, it is demonstrated that this interaction force is completely abrogated when T-cell signalling is blocked. As a result the potential of using optical trapping to interrogate cellular interactions at the single cell level without the need to introduce foreign bodies such as beads is clearly demonstrated. PMID:29220398

Empower multiplex cell and tissue-specific CRISPR-mediated gene manipulation with self-cleaving ribozymes and tRNA.

PubMed

Xu, Li; Zhao, Lixia; Gao, Yandi; Xu, Jing; Han, Renzhi

2017-03-17

Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) system has emerged in recent years as a highly efficient RNA-guided gene manipulation platform. Simultaneous editing or transcriptional activation/suppression of different genes becomes feasible with the co-delivery of multiple guide RNAs (gRNAs). Here, we report that multiple gRNAs linked with self-cleaving ribozymes and/or tRNA could be simultaneously expressed from a single U6 promoter to exert genome editing of dystrophin and myosin binding protein C3 in human and mouse cells. Moreover, this strategy allows the expression of multiple gRNAs for synergistic transcription activation of follistatin when used with catalytically inactive dCas9-VP64 or dCas9-p300core fusions. Finally, the gRNAs linked by the self-cleaving ribozymes and tRNA could be expressed from RNA polymerase type II (pol II) promoters such as generic CMV and muscle/heart-specific MHCK7. This is particularly useful for in vivo applications when the packaging capacity of recombinant adeno-associated virus is limited while tissue-specific delivery of gRNAs and Cas9 is desired. Taken together, this study provides a novel strategy to enable tissue-specific expression of more than one gRNAs for multiplex gene editing from a single pol II promoter. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector

PubMed Central

Kabadi, Ami M.; Ousterout, David G.; Hilton, Isaac B.; Gersbach, Charles A.

2014-01-01

Engineered DNA-binding proteins that manipulate the human genome and transcriptome have enabled rapid advances in biomedical research. In particular, the RNA-guided CRISPR/Cas9 system has recently been engineered to create site-specific double-strand breaks for genome editing or to direct targeted transcriptional regulation. A unique capability of the CRISPR/Cas9 system is multiplex genome engineering by delivering a single Cas9 enzyme and two or more single guide RNAs (sgRNAs) targeted to distinct genomic sites. This approach can be used to simultaneously create multiple DNA breaks or to target multiple transcriptional activators to a single promoter for synergistic enhancement of gene induction. To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method. Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types. PMID:25122746

Cellular manipulation and patterning using ferromagnetic nanowires

NASA Astrophysics Data System (ADS)

Hultgren, Anne

Ferromagnetic nanowires are demonstrated as an effective tool to apply forces to living cells. Both magnetic cell separations and the magnetic patterning of cells on a substrate will be accomplished through the use of cell-nanowire interactions as well as nanowire-magnetic field interactions. When introduced into cultures of NIH-3T3 cells, the nanowires are internalized by cells via the integrin-mediated adhesion pathway without inflicting any toxic effects on the cell cycle over the course of several days. In addition, the length of the nanowires was found to have an effect on the cell-nanowire interactions when the cells were dissociated from the tissue culture dish. To compare the effectiveness of the nanowires as a means of manipulating cells to the current technology which is based on superparamagnetic beads, magnetic cell separations were performed with electrodeposited Ni nanowires 350 nm in diameter and 5--35 mum long in field gradients of 80 T/m. Single-pass separations of NIH-3T3 cells bound to nanowires achieve up to 81% purity with 85% yield, a dramatic improvement over the 55% purity and 20% yield obtained with the beads. The yield for the separations were found to be dependent on the length of the nanowires, and was maximized when the length of the nanowires equaled the diameter of the cells. This dependence was exploited to perform a size-selective magnetic separation. Substrates containing arrays of micro-magnets, fabricated using photolithography, were placed in cell cultures. These micro-magnet arrays create regions of locally strong magnetic field gradients to trap nanowires in specific locations on the substrate. These substrates were used in conjunction with fluid flow and a weak, externally applied magnetic field to create and control patterns of cells bound to nanowires. Controlled isolation of heterogeneous pairs and groups of cells will enable the study of the biochemistry of cell-cell contacts.

Simple Perfusion Apparatus (SPA) for Manipulation, Tracking and Study of Oocytes and Embryos

PubMed Central

Angione, Stephanie L.; Oulhen, Nathalie; Brayboy, Lynae M.; Tripathi, Anubhav; Wessel, Gary M.

2016-01-01

Objective To develop and implement a device and protocol for oocyte analysis at a single cell level. The device must be capable of high resolution imaging, temperature control, perfusion of media, drugs, sperm, and immunolabeling reagents all at defined flow-rates. Each oocyte and resultant embryo must remain spatially separated and defined. Design Experimental laboratory study Setting University and Academic Center for reproductive medicine. Patients/Animals Women with eggs retrieved for ICSI cycles, adult female FVBN and B6C3F1 mouse strains, sea stars. Intervention Real-time, longitudinal imaging of oocytes following fluorescent labeling, insemination, and viability tests. Main outcome measure(s) Cell and embryo viability, immunolabeling efficiency, live cell endocytosis quantitation, precise metrics of fertilization and embryonic development. Results Single oocytes were longitudinally imaged following significant changes in media, markers, endocytosis quantitation, and development, all with supreme control by microfluidics. Cells remained viable, enclosed, and separate for precision measurements, repeatability, and imaging. Conclusions We engineered a simple device to load, visualize, experiment, and effectively record individual oocytes and embryos, without loss of cells. Prolonged incubation capabilities provide longitudinal studies without need for transfer and potential loss of cells. This simple perfusion apparatus (SPA) provides for careful, precise, and flexible handling of precious samples facilitating clinical in vitro fertilization approaches. PMID:25450296

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

PubMed Central

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. PMID:26727004

Microfluidic Cell-based Assays in Stem Cell and Other Rare Cell Type Research

DOE PAGES

Wu, Meiye

2015-03-23

Microfluidics is a technology defined by the engineered precise manipulation of minute amount of liquids through channels with dimensions in the micron scale. Much of microfluidic devices used for biomedical purposes are produced in the form of so called “lab-on-a-chip” format, where multiple steps of conventional biochemical analyses such as staining, washing, and signal collection are miniaturized and integrated into chips fabricated from polymer or glass. Cell-based microfluidic lab-on-achip technology provides some obvious advantages: 1) drastically reduced sample and reagent requirement, and 2) separation and detection with improved sensitivity due to fluid properties at the microscale, i.e. laminar flow. Basedmore » on these two advantages, the obvious place where microfluidic cell assays will provide the most benefit is wherescientists must gather much information from precious little sample. Stem cells and other precious cell types such as circulating tumor cells (CTCs), and rare immune subsets are the perfect match for microfluidic multiplex assays. The recent demonstration that multiple cellular changes such as surface receptor activation, protein translocation, long and short RNA, and DNA changes can all be extracted from intact single cells paves the way to systems level understanding of cellular states during development or disease. Finally, with the ability to preserve cell integrity in a microfluidic device during multiplexed analysis, one also preserves the single cell resolution, where information regarding the cell-to-cell heterogeneity during differentiation or response to stimuli is vitally important.« less

Manipulating molecular quantum states with classical metal atom inputs: demonstration of a single molecule NOR logic gate.

PubMed

Soe, We-Hyo; Manzano, Carlos; Renaud, Nicolas; de Mendoza, Paula; De Sarkar, Abir; Ample, Francisco; Hliwa, Mohamed; Echavarren, Antonio M; Chandrasekhar, Natarajan; Joachim, Christian

2011-02-22

Quantum states of a trinaphthylene molecule were manipulated by putting its naphthyl branches in contact with single Au atoms. One Au atom carries 1-bit of classical information input that is converted into quantum information throughout the molecule. The Au-trinaphthylene electronic interactions give rise to measurable energy shifts of the molecular electronic states demonstrating a NOR logic gate functionality. The NOR truth table of the single molecule logic gate was characterized by means of scanning tunnelling spectroscopy.

Ultrafast electric phase control of a single exciton qubit

NASA Astrophysics Data System (ADS)

Widhalm, Alex; Mukherjee, Amlan; Krehs, Sebastian; Sharma, Nandlal; Kölling, Peter; Thiede, Andreas; Reuter, Dirk; Förstner, Jens; Zrenner, Artur

2018-03-01

We report on the coherent phase manipulation of quantum dot excitons by electric means. For our experiments, we use a low capacitance single quantum dot photodiode which is electrically controlled by a custom designed SiGe:C BiCMOS chip. The phase manipulation is performed and quantified in a Ramsey experiment, where ultrafast transient detuning of the exciton energy is performed synchronous to double pulse π/2 ps laser excitation. We are able to demonstrate electrically controlled phase manipulations with magnitudes up to 3π within 100 ps which is below the dephasing time of the quantum dot exciton.

Resource reallocation does not influence estimates of pollen limitation or reproductive assurance in Clarkia xantiana subsp. parviflora (Onagraceae).

PubMed

Runquist, Ryan D Briscoe; Moeller, David A

2013-09-01

Studies of pollen limitation and the reproductive assurance value of selfing are important for examining the process of floral and mating system evolution in flowering plants. Recent meta-analyses have shown that common methods for measuring pollen limitation may often lead to biased estimates. Specifically, experiments involving single- or few-flower manipulations per plant tend to overestimate pollen limitation compared to those involving manipulations on most or all flowers per plant. Little previous work has explicitly tested for reallocation within individual systems using alternative methods and response variables. • We performed single-flower and whole-plant pollen supplementation and emasculation of flowers of Clarkia xantiana subsp. parviflora to estimate pollen limitation (PL) and reproductive assurance (RA). We compared levels of PL and RA using the following response variables: fruit set, seeds/flower, and seeds/plant. We also assessed the germination and viability of seeds to evaluate potential variation in pollen quality among treatments. • Autonomous selfing in Clarkia xantiana subsp. parviflora eliminates pollen limitation and provides reproductive assurance. Estimates from single-flower manipulations were not biased, closely resembling those from whole-plant manipulations. All three response variables followed the same pattern, but treatments were only significantly different for seeds/flower. Pollen quality, as indicated by seed viability, did not differ among treatments. • Partial plant manipulations provided reliable estimates of pollen limitation and reproductive assurance. These estimates were also unaffected by accounting for pollen quality. Although whole plant manipulations are desirable, this experiment demonstrates that in some systems partial plant manipulations can be used in studies where whole-plant manipulations are not feasible.

Host manipulation by cancer cells: Expectations, facts, and therapeutic implications.

PubMed

Tissot, Tazzio; Arnal, Audrey; Jacqueline, Camille; Poulin, Robert; Lefèvre, Thierry; Mery, Frédéric; Renaud, François; Roche, Benjamin; Massol, François; Salzet, Michel; Ewald, Paul; Tasiemski, Aurélie; Ujvari, Beata; Thomas, Frédéric

2016-03-01

Similar to parasites, cancer cells depend on their hosts for sustenance, proliferation and reproduction, exploiting the hosts for energy and resources, and thereby impairing their health and fitness. Because of this lifestyle similarity, it is predicted that cancer cells could, like numerous parasitic organisms, evolve the capacity to manipulate the phenotype of their hosts to increase their own fitness. We claim that the extent of this phenomenon and its therapeutic implications are, however, underappreciated. Here, we review and discuss what can be regarded as cases of host manipulation in the context of cancer development and progression. We elaborate on how acknowledging the applicability of these principles can offer novel therapeutic and preventive strategies. The manipulation of host phenotype by cancer cells is one more reason to adopt a Darwinian approach in cancer research. © 2016 WILEY Periodicals, Inc.

Near-field acoustic microbead trapping as remote anchor for single particle manipulation

NASA Astrophysics Data System (ADS)

Hwang, Jae Youn; Cheon, Dong Young; Shin, Hyunjune; Kim, Hyun Bin; Lee, Jungwoo

2015-05-01

We recently proposed an analytical model of a two-dimensional acoustic trapping of polystyrene beads in the ray acoustics regime, where a bead diameter is larger than the wavelength used. As its experimental validation, this paper demonstrates the transverse (or lateral) trapping of individual polystyrene beads in the near field of focused ultrasound. A 100 μm bead is immobilized on the central beam axis by a focused sound beam from a 30 MHz single element lithium niobate transducer, after being laterally displaced through hundreds of micrometers. Maximum displacement, a longest lateral distance at which a trapped bead can be directed towards the central axis, is thus measured over a discrete frequency range from 24 MHz to 36 MHz. The displacement data are found to be between 323.7 μm and 470.2 μm, depending on the transducer's driving frequency and input voltage amplitude. The experimental results are compared with their corresponding model values, and their relative errors lie between 0.9% and 3.9%. The results suggest that this remote maneuvering technique may be employed to manipulate individual cells through solid microbeads, provoking certain cellular reactions to localized mechanical disturbance without direct contact.

On-chip magnetically actuated robot with ultrasonic vibration for single cell manipulations.

PubMed

Hagiwara, Masaya; Kawahara, Tomohiro; Yamanishi, Yoko; Masuda, Taisuke; Feng, Lin; Arai, Fumihito

2011-06-21

This paper presents an innovative driving method for an on-chip robot actuated by permanent magnets in a microfluidic chip. A piezoelectric ceramic is applied to induce ultrasonic vibration to the microfluidic chip and the high-frequency vibration reduces the effective friction on the MMT significantly. As a result, we achieved 1.1 micrometre positioning accuracy of the microrobot, which is 100 times higher accuracy than without vibration. The response speed is also improved and the microrobot can be actuated with a speed of 5.5 mm s(-1) in 3 degrees of freedom. The novelty of the ultrasonic vibration appears in the output force as well. Contrary to the reduction of friction on the microrobot, the output force increased twice as much by the ultrasonic vibration. Using this high accuracy, high speed, and high power microrobot, swine oocyte manipulations are presented in a microfluidic chip.

Design of Fresnel Lens-Type Multi-Trapping Acoustic Tweezers

PubMed Central

Tu, You-Lin; Chen, Shih-Jui; Hwang, Yean-Ren

2016-01-01

In this paper, acoustic tweezers which use beam forming performed by a Fresnel zone plate are proposed. The performance has been demonstrated by finite element analysis, including the acoustic intensity, acoustic pressure, acoustic potential energy, gradient force, and particle distribution. The acoustic tweezers use an ultrasound beam produced by a lead zirconate titanate (PZT) transducer operating at 2.4 MHz and 100 Vpeak-to-peak in a water medium. The design of the Fresnel lens (zone plate) is based on air reflection, acoustic impedance matching, and the Fresnel half-wave band (FHWB) theory. This acoustic Fresnel lens can produce gradient force and acoustic potential wells that allow the capture and manipulation of single particles or clusters of particles. Simulation results strongly indicate a good trapping ability, for particles under 150 µm in diameter, in the minimum energy location. This can be useful for cell or microorganism manipulation. PMID:27886050

CMOS capacitive biosensors for highly sensitive biosensing applications.

PubMed

Chang, An-Yu; Lu, Michael S-C

2013-01-01

Magnetic microbeads are widely used in biotechnology and biomedical research for manipulation and detection of cells and biomolecules. Most lab-on-chip systems capable of performing manipulation and detection require external instruments to perform one of the functions, leading to increased size and cost. This work aims at developing an integrated platform to perform these two functions by implementing electromagnetic microcoils and capacitive biosensors on a CMOS (complementary metal oxide semiconductor) chip. Compared to most magnetic-type sensors, our detection method requires no externally applied magnetic fields and the associated fabrication is less complicated. In our experiment, microbeads coated with streptavidin were driven to the sensors located in the center of microcoils with functionalized anti-streptavidin antibody. Detection of a single microbead was successfully demonstrated using a capacitance-to-frequency readout. The average capacitance changes for the experimental and control groups were -5.3 fF and -0.2 fF, respectively.

Manipulation of Liquids Using Phased Array Generation of Acoustic Radiation Pressure

NASA Technical Reports Server (NTRS)

Oeftering, Richard C. (Inventor)

2000-01-01

A phased array of piezoelectric transducers is used to control and manipulate contained as well as uncontained fluids in space and earth applications. The transducers in the phased array are individually activated while being commonly controlled to produce acoustic radiation pressure and acoustic streaming. The phased array is activated to produce a single pulse, a pulse burst or a continuous pulse to agitate, segregate or manipulate liquids and gases. The phased array generated acoustic radiation pressure is also useful in manipulating a drop, a bubble or other object immersed in a liquid. The transducers can be arranged in any number of layouts including linear single or multi- dimensional, space curved and annular arrays. The individual transducers in the array are activated by a controller, preferably driven by a computer.

Optical tweezers and multiphoton microscopies integrated photonic tool for mechanical and biochemical cell processes studies

NASA Astrophysics Data System (ADS)

de Thomaz, A. A.; Faustino, W. M.; Fontes, A.; Fernandes, H. P.; Barjas-Castro, M. d. L.; Metze, K.; Giorgio, S.; Barbosa, L. C.; Cesar, C. L.

2007-09-01

The research in biomedical photonics is clearly evolving in the direction of the understanding of biological processes at the cell level. The spatial resolution to accomplish this task practically requires photonics tools. However, an integration of different photonic tools and a multimodal and functional approach will be necessary to access the mechanical and biochemical cell processes. This way we can observe mechanicaly triggered biochemical events or biochemicaly triggered mechanical events, or even observe simultaneously mechanical and biochemical events triggered by other means, e.g. electricaly. One great advantage of the photonic tools is its easiness for integration. Therefore, we developed such integrated tool by incorporating single and double Optical Tweezers with Confocal Single and Multiphoton Microscopies. This system can perform 2-photon excited fluorescence and Second Harmonic Generation microscopies together with optical manipulations. It also can acquire Fluorescence and SHG spectra of specific spots. Force, elasticity and viscosity measurements of stretched membranes can be followed by real time confocal microscopies. Also opticaly trapped living protozoas, such as leishmania amazonensis. Integration with CARS microscopy is under way. We will show several examples of the use of such integrated instrument and its potential to observe mechanical and biochemical processes at cell level.

Real Time Visualization and Manipulation of the Metastatic Trajectory ofBreast Cancer Cell

DTIC Science & Technology

2017-09-01

AWARD NUMBER: W81XWH-13-1-0173 TITLE: Real-Time Visualization and Manipulation of the Metastatic Trajectory of Breast Cancer Cells ...of this work was to engineer breast cancer cells to irreversibly alter the genome of nearby cells through exosomal transfer of Cre recombinase from...the cancer cells to surrounding cells . Our goal was to use this study to activate green fluorescent protein in the host reporter cells in the

Autofocusing and Polar Body Detection in Automated Cell Manipulation.

PubMed

Wang, Zenan; Feng, Chen; Ang, Wei Tech; Tan, Steven Yih Min; Latt, Win Tun

2017-05-01

Autofocusing and feature detection are two essential processes for performing automated biological cell manipulation tasks. In this paper, we have introduced a technique capable of focusing on a holding pipette and a mammalian cell under a bright-field microscope automatically, and a technique that can detect and track the presence and orientation of the polar body of an oocyte that is rotated at the tip of a micropipette. Both algorithms were evaluated by using mouse oocytes. Experimental results show that both algorithms achieve very high success rates: 100% and 96%. As robust and accurate image processing methods, they can be widely applied to perform various automated biological cell manipulations.

Manipulation of immune system via immortal bone marrow stem cells.

PubMed

Ruedl, Christiane; Khameneh, Hanif Javanmard; Karjalainen, Klaus

2008-09-01

Extensive amplification of hematopoietic stem cells (HSCs) and their multipotent primitive progenitors (MPPs) in culture would greatly benefit not only clinical transplantation but also provide a potential tool to manipulate all cellular lineages derived from these cells for gene therapy and experimental purposes. Here, we demonstrate that mouse bone marrow cultures containing cells engineered to over-express NUP98-HOXB4 fusion protein support self-renewal of physiologically normal HSC and MPP for several weeks leading practically to their unlimited expansion. This allows time consuming and cumulative in vitro experimental manipulations without sacrificing their ability to differentiate in vivo or in vitro to any hematopoietic lineage.

Stem cell maintenance by manipulating signaling pathways: past, current and future

PubMed Central

Chen, Xi; Ye, Shoudong; Ying, Qi-Long

2015-01-01

Pluripotent stem cells only exist in a narrow window during early embryonic development, whereas multipotent stem cells are abundant throughout embryonic development and are retainedin various adult tissues and organs. While pluripotent stem cell lines have been established from several species, including mouse, rat, and human, it is still challenging to establish stable multipotent stem cell lines from embryonic or adult tissues. Based on current knowledge, we anticipate that by manipulating extrinsic and intrinsic signaling pathways, most if not all types of stem cells can be maintained in a long-term culture. In this article, we summarize current culture conditions established for the long-term maintenance of authentic pluripotent and multipotent stem cells and the signaling pathways involved. We also discuss the general principles of stem cell maintenance and propose several strategies on the establishment of novel stem cell lines through manipulation of signaling pathways. [BMB Reports 2015; 48(12): 668-676] PMID:26497581

Drug-DNA interactions at single molecule level: A view with optical tweezers

NASA Astrophysics Data System (ADS)

Paramanathan, Thayaparan

Studies of small molecule--DNA interactions are essential for developing new drugs for challenging diseases like cancer and HIV. The main idea behind developing these molecules is to target and inhibit the reproduction of the tumor cells and infected cells. We mechanically manipulate single DNA molecule using optical tweezers to investigate two molecules that have complex and multiple binding modes. Mononuclear ruthenium complexes have been extensively studied as a test for rational drug design. Potential drug candidates should have high affinity to DNA and slow dissociation kinetics. To achieve this, motifs of the ruthenium complexes are altered. Our collaborators designed a dumb-bell shaped binuclear ruthenium complex that can only intercalate DNA by threading through its bases. Studying the binding properties of this complex in bulk studies took hours. By mechanically manipulating a single DNA molecule held with optical tweezers, we lower the barrier to thread and make it fast compared to the bulk experiments. Stretching single DNA molecules with different concentration of drug molecules and holding it at a constant force allows the binding to reach equilibrium. By this we can obtain the equilibrium fractional ligand binding and length of DNA at saturated binding. Fitting these results yields quantitative measurements of the binding thermodynamics and kinetics of this complex process. The second complex discussed in this study is Actinomycin D (ActD), a well studied anti-cancer agent that is used as a prototype for developing new generations of drugs. However, the biophysical basis of its activity is still unclear. Because ActD is known to intercalate double stranded DNA (dsDNA), it was assumed to block replication by stabilizing dsDNA in front of the replication fork. However, recent studies have shown that ActD binds with even higher affinity to imperfect duplexes and some sequences of single stranded DNA (ssDNA). We directly measure the on and off rates by stretching the DNA molecule to a certain force and holding it at constant force while adding the drug and then while washing off the drug. Our finding resolves the long lasting controversy of ActD binding modes, clearly showing that both the dsDNA binding and ssDNA binding converge to the same single mode. The result supports the hypothesis that the primary characteristic of ActD that contributes to its biological activity is its ability to inhibit cellular replication by binding to transcription bubbles and causing cell death.

In situ cell manipulation through enzymatic hydrogel photopatterning

NASA Astrophysics Data System (ADS)

Mosiewicz, Katarzyna A.; Kolb, Laura; van der Vlies, André J.; Martino, Mikaël M.; Lienemann, Philipp S.; Hubbell, Jeffrey A.; Ehrbar, Martin; Lutolf, Matthias P.

2013-11-01

The physicochemical properties of hydrogels can be manipulated in both space and time through the controlled application of a light beam. However, methods for hydrogel photopatterning either fail to maintain the bioactivity of fragile proteins and are thus limited to short peptides, or have been used in hydrogels that often do not support three-dimensional (3D) cell growth. Here, we show that the 3D invasion of primary human mesenchymal stem cells can be spatiotemporally controlled by micropatterning the hydrogel with desired extracellular matrix (ECM) proteins and growth factors. A peptide substrate of activated transglutaminase factor XIII (FXIIIa)—a key ECM crosslinking enzyme—is rendered photosensitive by masking its active site with a photolabile cage group. Covalent incorporation of the caged FXIIIa substrate into poly(ethylene glycol) hydrogels and subsequent laser-scanning lithography affords highly localized biomolecule tethering. This approach for the 3D manipulation of cells within gels should open up avenues for the study and manipulation of cell signalling.

Cell type-specific manipulation with GFP-dependent Cre recombinase.

PubMed

Tang, Jonathan C Y; Rudolph, Stephanie; Dhande, Onkar S; Abraira, Victoria E; Choi, Seungwon; Lapan, Sylvain W; Drew, Iain R; Drokhlyansky, Eugene; Huberman, Andrew D; Regehr, Wade G; Cepko, Constance L

2015-09-01

There are many transgenic GFP reporter lines that allow the visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. We developed a method that exploits GFP for gene manipulation, Cre recombinase dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, which led to effective recombination selectively in GFP-labeled cells. Furthermore, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP(+) cells, we found that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins.

Role of competition between polarity sites in establishing a unique front

PubMed Central

Wu, Chi-Fang; Chiou, Jian-Geng; Minakova, Maria; Woods, Benjamin; Tsygankov, Denis; Zyla, Trevin R; Savage, Natasha S; Elston, Timothy C; Lew, Daniel J

2015-01-01

Polarity establishment in many cells is thought to occur via positive feedback that reinforces even tiny asymmetries in polarity protein distribution. Cdc42 and related GTPases are activated and accumulate in a patch of the cortex that defines the front of the cell. Positive feedback enables spontaneous polarization triggered by stochastic fluctuations, but as such fluctuations can occur at multiple locations, how do cells ensure that they make only one front? In polarizing cells of the model yeast Saccharomyces cerevisiae, positive feedback can trigger growth of several Cdc42 clusters at the same time, but this multi-cluster stage rapidly evolves to a single-cluster state, which then promotes bud emergence. By manipulating polarity protein dynamics, we show that resolution of multi-cluster intermediates occurs through a greedy competition between clusters to recruit and retain polarity proteins from a shared intracellular pool. DOI: http://dx.doi.org/10.7554/eLife.11611.001 PMID:26523396

Investigation of HIV-1 infected and uninfected cells using the optical trapping technique

NASA Astrophysics Data System (ADS)

Ombinda-Lemboumba, S.; Malabi, R.; Lugongolo, M. Y.; Thobakgale, S. L.; Manoto, S.; Mthunzi-Kufa, P.

2017-02-01

Optical trapping has emerged as an essential tool for manipulating single biological material and performing sophisticated spectroscopy analysis on individual cell. The optical trapping technique has been used to grab and immobilize cells from a tightly focused laser beam emitted through a high numerical aperture objective lens. Coupling optical trapping with other technologies is possible and allows stable sample trapping, while also facilitating molecular, chemical and spectroscopic analysis. For this reason, we are exploring laser trapping combined with laser spectroscopy as a potential non-invasive method of interrogating individual cells with a high degree of specificity in terms of information generated. Thus, for the delivery of as much pathological information as possible, we use a home-build optical trapping and spectroscopy system for real time probing human immunodeficiency virus (HIV-1) infected and uninfected single cells. Briefly, our experimental rig comprises an infrared continuous wave laser at 1064 nm with power output of 1.5 W, a 100X high numerical aperture oil-immersion microscope objective used to capture and immobilise individual cell samples as well as an excitation source. Spectroscopy spectral patterns obtained by the 1064 nm laser beam excitation provide information on HIV-1 infected and uninfected cells. We present these preliminary findings which may be valuable for the development of an HIV-1 point of care detection system.

Ultrafast Magnetization Manipulation Using Single Femtosecond Light and Hot-Electron Pulses.

PubMed

Xu, Yong; Deb, Marwan; Malinowski, Grégory; Hehn, Michel; Zhao, Weisheng; Mangin, Stéphane

2017-11-01

Current-induced magnetization manipulation is a key issue for spintronic applications. This manipulation must be fast, deterministic, and nondestructive in order to function in device applications. Therefore, single- electronic-pulse-driven deterministic switching of the magnetization on the picosecond timescale represents a major step toward future developments of ultrafast spintronic systems. Here, the ultrafast magnetization dynamics in engineered Gd x [FeCo] 1- x -based structures are studied to compare the effect of femtosecond laser and hot-electron pulses. It is demonstrated that a single femtosecond hot-electron pulse causes deterministic magnetization reversal in either Gd-rich and FeCo-rich alloys similarly to a femtosecond laser pulse. In addition, it is shown that the limiting factor of such manipulation for perpendicular magnetized films arises from the formation of a multidomain state due to dipolar interactions. By performing time-resolved measurements under various magnetic fields, it is demonstrated that the same magnetization dynamics are observed for both light and hot-electron excitation, and that the full magnetization reversal takes place within 40 ps. The efficiency of the ultrafast current-induced magnetization manipulation is enhanced due to the ballistic transport of hot electrons before reaching the GdFeCo magnetic layer. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reprogramming cellular identity for regenerative medicine

PubMed Central

Cherry, Anne B.C.; Daley, George Q.

2012-01-01

The choreographed development of over 200 distinct differentiated cell types from a single zygote is a complex and poorly understood process. Whereas development leads unidirectionally towards more restricted cell fates, recent work in cellular reprogramming has proven that striking conversions of one cellular identity into another can be engineered, promising countless applications in biomedical research and paving the way for modeling disease with patient-derived stem cells. To date, there has been little discussion of which disease models are likely to be most informative. We here review evidence demonstrating that because environmental influences and epigenetic signatures are largely erased during reprogramming, patient-specific models of diseases with strong genetic bases and high penetrance are likely to prove most informative in the near term. However, manipulating in vitro culture conditions may ultimately enable cell-based models to recapitulate gene-environment interactions. Here, we discuss the implications of the new reprogramming paradigm in biomedicine and outline how reprogramming of cell identities is enhancing our understanding of cell differentiation and prospects for cellular therapies and in vivo regeneration. PMID:22424223

Shaping magnetic fields to direct therapy to ears and eyes.

PubMed

Shapiro, B; Kulkarni, S; Nacev, A; Sarwar, A; Preciado, D; Depireux, D A

2014-07-11

Magnetic fields have the potential to noninvasively direct and focus therapy to disease targets. External magnets can apply forces on drug-coated magnetic nanoparticles, or on living cells that contain particles, and can be used to manipulate them in vivo. Significant progress has been made in developing and testing safe and therapeutic magnetic constructs that can be manipulated by magnetic fields. However, we do not yet have the magnet systems that can then direct those constructs to the right places, in vivo, over human patient distances. We do not yet know where to put the external magnets, how to shape them, or when to turn them on and off to direct particles or magnetized cells-in blood, through tissue, and across barriers-to disease locations. In this article, we consider ear and eye disease targets. Ear and eye targets are too deep and complex to be targeted by a single external magnet, but they are shallow enough that a combination of magnets may be able to direct therapy to them. We focus on how magnetic fields should be shaped (in space and time) to direct magnetic constructs to ear and eye targets.

Novel method for the high-throughput processing of slides for the comet assay

PubMed Central

Karbaschi, Mahsa; Cooke, Marcus S.

2014-01-01

Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. “Scoring”, or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure. PMID:25425241

Novel method for the high-throughput processing of slides for the comet assay.

PubMed

Karbaschi, Mahsa; Cooke, Marcus S

2014-11-26

Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. "Scoring", or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure.

Polymer Solar Cells with 90% External Quantum Efficiency Featuring an Ideal Light- and Charge-Manipulation Layer.

PubMed

Chen, Jing-De; Li, Yan-Qing; Zhu, Jingshuai; Zhang, Qianqian; Xu, Rui-Peng; Li, Chi; Zhang, Yue-Xing; Huang, Jing-Sheng; Zhan, Xiaowei; You, Wei; Tang, Jian-Xin

2018-03-01

Rapid progress in the power conversion efficiency (PCE) of polymer solar cells (PSEs) is beneficial from the factors that match the irradiated solar spectrum, maximize incident light absorption, and reduce photogenerated charge recombination. To optimize the device efficiency, a nanopatterned ZnO:Al 2 O 3 composite film is presented as an efficient light- and charge-manipulation layer (LCML). The Al 2 O 3 shells on the ZnO nanoparticles offer the passivation effect that allows optimal electron collection by suppressing charge-recombination loss. Both the increased refractive index and the patterned deterministic aperiodic nanostructure in the ZnO:Al 2 O 3 LCML cause broadband light harvesting. Highly efficient single-junction PSCs for different binary blends are obtained with a peak external quantum efficiency of up to 90%, showing certified PCEs of 9.69% and 13.03% for a fullerene blend of PTB7:PC 71 BM and a nonfullerene blend, FTAZ:IDIC, respectively. Because of the substantial increase in efficiency, this method unlocks the full potential of the ZnO:Al 2 O 3 LCML toward future photovoltaic applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Double-layered cell transfer technology for bone regeneration

PubMed Central

Akazawa, Keiko; Iwasaki, Kengo; Nagata, Mizuki; Yokoyama, Naoki; Ayame, Hirohito; Yamaki, Kazumasa; Tanaka, Yuichi; Honda, Izumi; Morioka, Chikako; Kimura, Tsuyoshi; Komaki, Motohiro; Kishida, Akio; Izumi, Yuichi; Morita, Ikuo

2016-01-01

For cell-based medicine, to mimic in vivo cellular localization, various tissue engineering approaches have been studied to obtain a desirable arrangement of cells on scaffold materials. We have developed a novel method of cell manipulation called “cell transfer technology”, enabling the transfer of cultured cells onto scaffold materials, and controlling cell topology. Here we show that using this technique, two different cell types can be transferred onto a scaffold surface as stable double layers or in patterned arrangements. Various combinations of adherent cells were transferred to a scaffold, amniotic membrane, in overlapping bilayers (double-layered cell transfer), and transferred cells showed stability upon deformations of the material including folding and trimming. Transplantation of mesenchymal stem cells from periodontal ligaments (PDLSC) and osteoblasts, using double-layered cell transfer significantly enhanced bone formation, when compared to single cell type transplantation. Our findings suggest that this double-layer cell transfer is useful to produce a cell transplantation material that can bear two cell layers. Moreover, the transplantation of an amniotic membrane with PDLSCs/osteoblasts by cell transfer technology has therapeutic potential for bone defects. We conclude that cell transfer technology provides a novel and unique cell transplantation method for bone regeneration. PMID:27624174

Rapid and efficient genetic engineering of both wild type and axenic strains of Dictyostelium discoideum

PubMed Central

Knecht, David A.; Silale, Augustinas; Traynor, David; Williams, Thomas D.; Thomason, Peter A.; Insall, Robert H.; Chubb, Jonathan R.; Kay, Robert R.; Veltman, Douwe M.

2018-01-01

Dictyostelium has a mature technology for molecular-genetic manipulation based around transfection using several different selectable markers, marker re-cycling, homologous recombination and insertional mutagenesis, all supported by a well-annotated genome. However this technology is optimized for mutant, axenic cells that, unlike non-axenic wild type, can grow in liquid medium. There is a pressing need for methods to manipulate wild type cells and ones with defects in macropinocytosis, neither of which can grow in liquid media. Here we present a panel of molecular genetic techniques based on the selection of Dictyostelium transfectants by growth on bacteria rather than liquid media. As well as extending the range of strains that can be manipulated, these techniques are faster than conventional methods, often giving usable numbers of transfected cells within a few days. The methods and plasmids described here allow efficient transfection with extrachromosomal vectors, as well as chromosomal integration at a ‘safe haven’ for relatively uniform cell-to-cell expression, efficient gene knock-in and knock-out and an inducible expression system. We have thus created a complete new system for the genetic manipulation of Dictyostelium cells that no longer requires cell feeding on liquid media. PMID:29847546

On-Chip Magnetic Platform for Single-Particle Manipulation with Integrated Electrical Feedback.

PubMed

Monticelli, Marco; Torti, Andrea; Cantoni, Matteo; Petti, Daniela; Albisetti, Edoardo; Manzin, Alessandra; Guerriero, Erica; Sordan, Roman; Gervasoni, Giacomo; Carminati, Marco; Ferrari, Giorgio; Sampietro, Marco; Bertacco, Riccardo

2016-02-17

Methods for the manipulation of single magnetic particles have become very interesting, in particular for in vitro biological studies. Most of these studies require an external microscope to provide the operator with feedback for controlling the particle motion, thus preventing the use of magnetic particles in high-throughput experiments. In this paper, a simple and compact system with integrated electrical feedback is presented, implementing in the very same device both the manipulation and detection of the transit of single particles. The proposed platform is based on zig-zag shaped magnetic nanostructures, where transverse magnetic domain walls are pinned at the corners and attract magnetic particles in suspension. By applying suitable external magnetic fields, the domain walls move to the nearest corner, thus causing the step by step displacement of the particles along the nanostructure. The very same structure is also employed for detecting the bead transit. Indeed, the presence of the magnetic particle in suspension over the domain wall affects the depinning field required for its displacement. This characteristic field can be monitored through anisotropic magnetoresistance measurements, thus implementing an integrated electrical feedback of the bead transit. In particular, the individual manipulation and detection of single 1-μm sized beads is demonstrated. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Testing inferior colliculus neurons for selectivity to the rate or duration of frequency modulated sweeps

NASA Astrophysics Data System (ADS)

Faure, Paul A.; Morrison, James A.; Valdizón-Rodríguez, Roberto

2018-05-01

Here we propose a method for testing how the responses of so-called "FM duration-tuned neurons (DTNs)" encode temporal properties of frequency modulated (FM) sweeps to determine if the responses of so-called "FM duration-tuned neurons (DTNs)" are tuned to FM rate or FM duration. Based on previous studies it was unclear if the responses of "FM DTNs" were tuned to signal duration, like pure-tone DTNs, or FM sweep rate. We tested this using single-unit extracellular recording in the inferior colliculus (IC) of the big brown bat (Eptesicus fuscus). We presented IC cells with linear FM sweeps that were varied in FM center frequency (CEF) and spectral bandwidth (BW) to measure the FM rate tuning responses of a cell. We also varied FM signal duration to measure the best duration (BD) and temporal BW of duration tuning of a cell. We then doubled (and halved) the best FM BW, while keeping the CEF constant, and remeasured the BD and temporal BW of duration tuning with FM bandwidth manipulated signals. We reasoned that the range of excitatory signal durations should not change in a true FM DTN whose responses are tuned to signal duration; however, when stimulated with bandwidth manipulated FM sounds the range of excitatory signal durations should predictably vary in a FM rate-tuned cell. Preliminary data indicate that our stimulus paradigm can disambiguate whether the evoked responses of an IC neuron are FM sweep rate tuned or FM duration tuned.

Remote Learning for the Manipulation and Control of Robotic Cells

ERIC Educational Resources Information Center

Goldstain, Ofir; Ben-Gal, Irad; Bukchin, Yossi

2007-01-01

This work proposes an approach to remote learning of robotic cells based on internet and simulation tools. The proposed approach, which integrates remote-learning and tele-operation into a generic scheme, is designed to enable students and developers to set-up and manipulate a robotic cell remotely. Its implementation is based on a dedicated…

Move, Stop, Learn: Illustrating Mitosis through Stop-Motion Animation

ERIC Educational Resources Information Center

Kamp, Brandi L.; Deaton, Cynthia C. M.

2013-01-01

Learning about microscopic things, such as cells, can often be mundane to students because they are not able to see or manipulate what they are learning about. Students often recall learning about cell division through memorization--thus they find it tedious and dull. Few opportunities exist that allow students to explore and manipulate cells or…

DNA methylation regulates neurophysiological spatial representation in memory formation

PubMed Central

Roth, Eric D.; Roth, Tania L.; Money, Kelli M.; SenGupta, Sonda; Eason, Dawn E.; Sweatt, J. David

2015-01-01

Epigenetic mechanisms including altered DNA methylation are critical for altered gene transcription subserving synaptic plasticity and the retention of learned behavior. Here we tested the idea that one role for activity-dependent altered DNA methylation is stabilization of cognition-associated hippocampal place cell firing in response to novel place learning. We observed that a behavioral protocol (spatial exploration of a novel environment) known to induce hippocampal place cell remapping resulted in alterations of hippocampal Bdnf DNA methylation. Further studies using neurophysiological in vivo single unit recordings revealed that pharmacological manipulations of DNA methylation decreased long-term but not short-term place field stability. Together our data highlight a role for DNA methylation in regulating neurophysiological spatial representation and memory formation. PMID:25960947

Fuel cell crimp-resistant cooling device with internal coil

NASA Technical Reports Server (NTRS)

Wittel, deceased, Charles F. (Inventor)

1986-01-01

A cooling assembly for fuel cells having a simplified construction whereby coolant is efficiently circulated through a conduit arranged in serpentine fashion in a channel within a member of such assembly. The channel is adapted to cradle a flexible, chemically inert, conformable conduit capable of manipulation into a variety of cooling patterns without crimping or otherwise restricting of coolant flow. The conduit, when assembled with the member, conforms into intimate contact with the member for good thermal conductivity. The conduit is non-corrodible and can be constructed as a single, manifold-free, continuous coolant passage means having only one inlet and one outlet. The conduit has an internal coil means which enables it to be bent in small radii without crimping.

Force-Mediating Magnetic Nanoparticles to Engineer Neuronal Cell Function

PubMed Central

Gahl, Trevor J.; Kunze, Anja

2018-01-01

Cellular processes like membrane deformation, cell migration, and transport of organelles are sensitive to mechanical forces. Technically, these cellular processes can be manipulated through operating forces at a spatial precision in the range of nanometers up to a few micrometers through chaperoning force-mediating nanoparticles in electrical, magnetic, or optical field gradients. But which force-mediating tool is more suitable to manipulate cell migration, and which, to manipulate cell signaling? We review here the differences in forces sensation to control and engineer cellular processes inside and outside the cell, with a special focus on neuronal cells. In addition, we discuss technical details and limitations of different force-mediating approaches and highlight recent advancements of nanomagnetics in cell organization, communication, signaling, and intracellular trafficking. Finally, we give suggestions about how force-mediating nanoparticles can be used to our advantage in next-generation neurotherapeutic devices. PMID:29867315

Manipulating biological agents and cells in micro-scale volumes for applications in medicine

PubMed Central

Tasoglu, Savas; Gurkan, Umut Atakan; Wang, ShuQi

2013-01-01

Recent technological advances provide new tools to manipulate cells and biological agents in micro/nano-liter volumes. With precise control over small volumes, the cell microenvironment and other biological agents can be bioengineered; interactions between cells and external stimuli can be monitored; and the fundamental mechanisms such as cancer metastasis and stem cell differentiation can be elucidated. Technological advances based on the principles of electrical, magnetic, chemical, optical, acoustic, and mechanical forces lead to novel applications in point-of-care diagnostics, regenerative medicine, in vitro drug testing, cryopreservation, and cell isolation/purification. In this review, we first focus on the underlying mechanisms of emerging examples for cell manipulation in small volumes targeting applications such as tissue engineering. Then, we illustrate how these mechanisms impact the aforementioned biomedical applications, discuss the associated challenges, and provide perspectives for further development. PMID:23575660

Force-Mediating Magnetic Nanoparticles to Engineer Neuronal Cell Function.

PubMed

Gahl, Trevor J; Kunze, Anja

2018-01-01

Cellular processes like membrane deformation, cell migration, and transport of organelles are sensitive to mechanical forces. Technically, these cellular processes can be manipulated through operating forces at a spatial precision in the range of nanometers up to a few micrometers through chaperoning force-mediating nanoparticles in electrical, magnetic, or optical field gradients. But which force-mediating tool is more suitable to manipulate cell migration, and which, to manipulate cell signaling? We review here the differences in forces sensation to control and engineer cellular processes inside and outside the cell, with a special focus on neuronal cells. In addition, we discuss technical details and limitations of different force-mediating approaches and highlight recent advancements of nanomagnetics in cell organization, communication, signaling, and intracellular trafficking. Finally, we give suggestions about how force-mediating nanoparticles can be used to our advantage in next-generation neurotherapeutic devices.

Multifunctional, inexpensive, and reusable nanoparticle-printed biochip for cell manipulation and diagnosis

PubMed Central

Esfandyarpour, Rahim; DiDonato, Matthew J.; Yang, Yuxin; Durmus, Naside Gozde; Harris, James S.; Davis, Ronald W.

2017-01-01

Isolation and characterization of rare cells and molecules from a heterogeneous population is of critical importance in diagnosis of common lethal diseases such as malaria, tuberculosis, HIV, and cancer. For the developing world, point-of-care (POC) diagnostics design must account for limited funds, modest public health infrastructure, and low power availability. To address these challenges, here we integrate microfluidics, electronics, and inkjet printing to build an ultra–low-cost, rapid, and miniaturized lab-on-a-chip (LOC) platform. This platform can perform label-free and rapid single-cell capture, efficient cellular manipulation, rare-cell isolation, selective analytical separation of biological species, sorting, concentration, positioning, enumeration, and characterization. The miniaturized format allows for small sample and reagent volumes. By keeping the electronics separate from microfluidic chips, the former can be reused and device lifetime is extended. Perhaps most notably, the device manufacturing is significantly less expensive, time-consuming, and complex than traditional LOC platforms, requiring only an inkjet printer rather than skilled personnel and clean-room facilities. Production only takes 20 min (vs. up to weeks) and $0.01—an unprecedented cost in clinical diagnostics. The platform works based on intrinsic physical characteristics of biomolecules (e.g., size and polarizability). We demonstrate biomedical applications and verify cell viability in our platform, whose multiplexing and integration of numerous steps and external analyses enhance its application in the clinic, including by nonspecialists. Through its massive cost reduction and usability we anticipate that our platform will enable greater access to diagnostic facilities in developed countries as well as POC diagnostics in resource-poor and developing countries. PMID:28167769

Totipotency, Pluripotency and Nuclear Reprogramming

NASA Astrophysics Data System (ADS)

Mitalipov, Shoukhrat; Wolf, Don

Mammalian development commences with the totipotent zygote which is capable of developing into all the specialized cells that make up the adult animal. As development unfolds, cells of the early embryo proliferate and differentiate into the first two lineages, the pluripotent inner cell mass and the trophectoderm. Pluripotent cells can be isolated, adapted and propagated indefinitely in vitro in an undifferentiated state as embryonic stem cells (ESCs). ESCs retain their ability to differentiate into cells representing the three major germ layers: endoderm, mesoderm or ectoderm or any of the 200+ cell types present in the adult body. Since many human diseases result from defects in a single cell type, pluripotent human ESCs represent an unlimited source of any cell or tissue type for replacement therapy thus providing a possible cure for many devastating conditions. Pluripotent cells resembling ESCs can also be derived experimentally by the nuclear reprogramming of somatic cells. Reprogrammed somatic cells may have an even more important role in cell replacement therapies since the patient's own somatic cells can be used for reprogramming thereby eliminating immune based rejection of transplanted cells. In this review, we summarize two major approaches to reprogramming: (1) somatic cell nuclear transfer and (2) direct reprogramming using genetic manipulations.

Effective cell trapping using PDMS microspheres in an acoustofluidic chip.

PubMed

Yin, Di; Xu, Gangwei; Wang, Mengyuan; Shen, Mingwu; Xu, Tiegang; Zhu, Xiaoyue; Shi, Xiangyang

2017-09-01

We present a facile particle-based cell manipulation method using acoustic radiation forces. In this work, we selected several representative particles including poly(lactic-co-glycolic acid) (PLGA) microspheres, silica-coated magnetic microbeads, polydimethylsiloxane (PDMS) microspheres and investigated the responses of these particle systems to ultrasonic standing waves (USWs) in a microfluidic chip. We show that depending on the nature (positive or negative acoustic contrast factors) of the particles, these particle systems display different alignment behaviors along the microfluidic channel under USWs. Specifically, PLGA microspheres and silica-coated magnetic microbeads are able to be aligned in the middle of the microfluidic channel, while PDMS microspheres are translocated to the side walls of the channel, which is beneficial for cell trapping and manipulation. Further results demonstrate that the functional PDMS microspheres with a negative acoustic contrast factor can be used to trap cells to the pressure antinodes in the acoustofluidic chip. Cell viability tests reveal that the ultrasonic manipulation does not exert any harmful effect to the cells. This acoustic-based particle and cell manipulation technique may hold a great promise for the development of rapid, noninvasive, continuous assays for detecting of cells and separation of biological samples. Copyright © 2017 Elsevier B.V. All rights reserved.

Controlled permeation of cell membrane by single bubble acoustic cavitation

PubMed Central

Zhou, Y.; Yang, K.; Cui, J.; Ye, J. Y.; Deng, C. X.

2011-01-01

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustic, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency (7.44 MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5 MHz) ultrasound pulse (duration 13.3 or 40 µs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d = 0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106 ± 0.032 µm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 µs), and increased to 0.171 ± 0.030 µm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 µm (n=112) for a pulse duration of 40 µs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level. PMID:21945682

Redundant arm control in a supervisory and shared control system

NASA Technical Reports Server (NTRS)

Backes, Paul G.; Long, Mark K.

1992-01-01

The Extended Task Space Control approach to robotic operations based on manipulator behaviors derived from task requirements is described. No differentiation between redundant and non-redundant robots is made at the task level. The manipulation task behaviors are combined into a single set of motion commands. The manipulator kinematics are used subsequently in mapping motion commands into actuator commands. Extended Task Space Control is applied to a Robotics Research K-1207 seven degree-of-freedom manipulator in a supervisory telerobot system as an example.

Genetic improvement of leaf photosynthesis and intrinsic water use efficiency in C3 plants: Why so much little success?

PubMed

Flexas, J

2016-10-01

There is an urgent need for simultaneously increasing photosynthesis/yields and water use efficiency (WUE) in C3 crops. Potentially, this can be achieved by genetic manipulation of the key traits involved. However, despite significant efforts in the past two decades very limited success has been achieved. Here I argue that this is mostly due to the fact that single gene/single trait approaches have been used thus far. Photosynthesis models demonstrate that only limited improving of photosynthesis can be expected by large improvements of any of its single limiting factors, i.e. stomatal conductance, mesophyll conductance, and the biochemical capacity for photosynthesis, the latter co-limited by Rubisco and the orchestrated activity of thylakoid electron transport and the Calvin cycle enzymes. Accordingly, only limited improvements of photosynthesis have been obtained by genetic manipulation of any of these single factors. In addition, improving photosynthesis by genetic manipulation in general reduced WUE, and vice-versa, and in many cases pleiotropic effects appear that cancel out some of the expected benefits. I propose that success in genetic manipulation for simultaneous improvement of photosynthesis and WUE efficiency may take longer than suggested in previous reports, and that it can be achieved only by joint projects addressing multi-gene manipulation for simultaneous alterations of all the limiting factors of photosynthesis, including the often neglected phloem capacity for loading and transport the expected surplus of carbohydrates in plants with improved photosynthesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Design of a Single-Cell Positioning Controller Using Electroosmotic Flow and Image Processing

PubMed Central

Ay, Chyung; Young, Chao-Wang; Chen, Jhong-Yin

2013-01-01

The objective of the current research was not only to provide a fast and automatic positioning platform for single cells, but also improved biomolecular manipulation techniques. In this study, an automatic platform for cell positioning using electroosmotic flow and image processing technology was designed. The platform was developed using a PCI image acquisition interface card for capturing images from a microscope and then transferring them to a computer using human-machine interface software. This software was designed by the Laboratory Virtual Instrument Engineering Workbench, a graphical language for finding cell positions and viewing the driving trace, and the fuzzy logic method for controlling the voltage or time of an electric field. After experiments on real human leukemic cells (U-937), the success of the cell positioning rate achieved by controlling the voltage factor reaches 100% within 5 s. A greater precision is obtained when controlling the time factor, whereby the success rate reaches 100% within 28 s. Advantages in both high speed and high precision are attained if these two voltage and time control methods are combined. The control speed with the combined method is about 5.18 times greater than that achieved by the time method, and the control precision with the combined method is more than five times greater than that achieved by the voltage method. PMID:23698272

Single and multi-band electromagnetic induced transparency-like metamaterials with coupled split ring resonators

NASA Astrophysics Data System (ADS)

Bagci, Fulya; Akaoglu, Baris

2017-08-01

We present a metamaterial configuration exhibiting single and multi-band electromagnetic induced transparency (EIT)-like properties. The unit cell of the single band EIT-like metamaterial consists of a multi-split ring resonator surrounded by a split ring resonator. The multi-split ring resonator acts as a quasi-dark or dark resonator, depending on the polarization of the incident wave, and the split ring resonator serves as the bright resonator. Combination of these two resonators results in a single band EIT-like transmission inside the stop band. EIT-like transmission phenomenon is also clearly observed in the measured transmission spectrum at almost the same frequencies for vertical and horizontal polarized waves, and the numerical results are verified for normal incidence. Moreover, multi-band transmission windows are created within a wide band by combining the two slightly different single band EIT-like metamaterial unit cells that exhibit two different coupling strengths inside a supercell configuration. Group indices as high as 123 for single band and 488 for tri-band transmission, accompanying with high transmission rates (over 80%), are achieved, rendering the metamaterial very suitable for multi-band slow light applications. It is shown that the group delay of the propagating wave can be increased and dynamically controlled by changing the polarization angle. Multi-band EIT-like transmission is also verified experimentally, and a good agreement with simulations is obtained. The proposed novel methodology for obtaining multi-band EIT, which takes advantage of a supercell configuration by hosting slightly different configured unit cells, can be utilized for easily formation and manipulation of multi-band transmission windows inside a stop band.

Magnetic tagging of cell-derived microparticles: new prospects for imaging and manipulation of these mediators of biological information.

PubMed

Vats, Nidhi; Wilhelm, Claire; Rautou, Pierre-Emmanuel; Poirier-Quinot, Marie; Péchoux, Christine; Devue, Cécile; Boulanger, Chantal M; Gazeau, Florence

2010-07-01

Submicron membrane fragments termed microparticles (MPs), which are released by apoptotic or activated cells, are newly considered as vectors of biological information and actors of pathology development. We propose the tagging of MPs with magnetic nanoparticles as a new approach allowing imaging, manipulation and targeting of cell-derived MPs. MPs generated in vitro from human endothelial cells or isolated from atherosclerotic plaques were labeled using citrate-coated 8 nm iron-oxide nanoparticles. MPs were tagged with magnetic nanoparticles on their surface and detected as Annexin-V positive by flow cytometry. Labeled MPs could be mobilized, isolated and manipulated at a distance in a magnetic field gradient. Magnetic mobility of labeled MPs was quantified by micromagnetophoresis. Interactions of labeled MPs with endothelial cells could be triggered and modulated by magnetic guidance. Nanoparticles served as tracers at different scales: at the subcellular level by electron microscopy, at the cellular level by histology and at the macroscopic level by MRI. Magnetic labeling of biogenic MPs opens new prospects for noninvasive monitoring and distal manipulations of these biological effectors.

Preparation, Single-Molecule Manipulation, and Energy Transfer Investigation of a Polyfluorene-graft-DNA polymer.

PubMed

Madsen, Mikael; Christensen, Rasmus S; Krissanaprasit, Abhichart; Bakke, Mette R; Riber, Camilla F; Nielsen, Karina S; Zelikin, Alexander N; Gothelf, Kurt V

2017-08-04

Conjugated polymers have been intensively studied due to their unique optical and electronic properties combined with their physical flexibility and scalable bottom up synthesis. Although the bulk qualities of conjugated polymers have been extensively utilized in research and industry, the ability to handle and manipulate conjugated polymers at the nanoscale lacks significantly behind. Here, the toolbox for controlled manipulation of conjugated polymers was expanded through the synthesis of a polyfluorene-DNA graft-type polymer (poly(F-DNA)). The polymer possesses the characteristics associated with the conjugated polyfluorene backbone, but the protruding single-stranded DNA provides the material with an exceptional addressability. This study demonstrates controlled single-molecule patterning of poly(F-DNA), as well as energy transfer between two different polymer-DNA conjugates. Finally, highly efficient DNA-directed quenching of polyfluorene fluorescence was shown. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Coherent manipulation of a solid-state artificial atom with few photons.

PubMed

Giesz, V; Somaschi, N; Hornecker, G; Grange, T; Reznychenko, B; De Santis, L; Demory, J; Gomez, C; Sagnes, I; Lemaître, A; Krebs, O; Lanzillotti-Kimura, N D; Lanco, L; Auffeves, A; Senellart, P

2016-06-17

In a quantum network based on atoms and photons, a single atom should control the photon state and, reciprocally, a single photon should allow the coherent manipulation of the atom. Both operations require controlling the atom environment and developing efficient atom-photon interfaces, for instance by coupling the natural or artificial atom to cavities. So far, much attention has been drown on manipulating the light field with atomic transitions, recently at the few-photon limit. Here we report on the reciprocal operation and demonstrate the coherent manipulation of an artificial atom by few photons. We study a quantum dot-cavity system with a record cooperativity of 13. Incident photons interact with the atom with probability 0.95, which radiates back in the cavity mode with probability 0.96. Inversion of the atomic transition is achieved for 3.8 photons on average, showing that our artificial atom performs as if fully isolated from the solid-state environment.

Cell Type-Specific Manipulation with GFP-Dependent Cre Recombinase

PubMed Central

Tang, Jonathan C Y; Rudolph, Stephanie; Dhande, Onkar S; Abraira, Victoria E; Choi, Seungwon; Lapan, Sylvain; Drew, Iain R; Drokhlyansky, Eugene; Huberman, Andrew D; Regehr, Wade G; Cepko, Constance L

2016-01-01

Summary There are many transgenic GFP reporter lines that allow visualization of specific populations of cells. Using such lines for functional studies requires a method that transforms GFP into a molecule that enables genetic manipulation. Here we report the creation of a method that exploits GFP for gene manipulation, Cre Recombinase Dependent on GFP (CRE-DOG), a split component system that uses GFP and its derivatives to directly induce Cre/loxP recombination. Using plasmid electroporation and AAV viral vectors, we delivered CRE-DOG to multiple GFP mouse lines, leading to effective recombination selectively in GFP-labeled cells. Further, CRE-DOG enabled optogenetic control of these neurons. Beyond providing a new set of tools for manipulation of gene expression selectively in GFP+ cells, we demonstrate that GFP can be used to reconstitute the activity of a protein not known to have a modular structure, suggesting that this strategy might be applicable to a wide range of proteins. PMID:26258682

Photoelectrochemical modulation of neuronal activity with free-standing coaxial silicon nanowires

NASA Astrophysics Data System (ADS)

Parameswaran, Ramya; Carvalho-de-Souza, João L.; Jiang, Yuanwen; Burke, Michael J.; Zimmerman, John F.; Koehler, Kelliann; Phillips, Andrew W.; Yi, Jaeseok; Adams, Erin J.; Bezanilla, Francisco; Tian, Bozhi

2018-02-01

Optical methods for modulating cellular behaviour are promising for both fundamental and clinical applications. However, most available methods are either mechanically invasive, require genetic manipulation of target cells or cannot provide subcellular specificity. Here, we address all these issues by showing optical neuromodulation with free-standing coaxial p-type/intrinsic/n-type silicon nanowires. We reveal the presence of atomic gold on the nanowire surfaces, likely due to gold diffusion during the material growth. To evaluate how surface gold impacts the photoelectrochemical properties of single nanowires, we used modified quartz pipettes from a patch clamp and recorded sustained cathodic photocurrents from single nanowires. We show that these currents can elicit action potentials in primary rat dorsal root ganglion neurons through a primarily atomic gold-enhanced photoelectrochemical process.

Manufacturing models permitting roll out/scale out of clinically led autologous cell therapies: regulatory and scientific challenges for comparability.

PubMed

Hourd, Paul; Ginty, Patrick; Chandra, Amit; Williams, David J

2014-08-01

Manufacturing of more-than-minimally manipulated autologous cell therapies presents a number of unique challenges driven by complex supply logistics and the need to scale out production to multiple manufacturing sites or near the patient within hospital settings. The existing regulatory structure in Europe and the United States imposes a requirement to establish and maintain comparability between sites. Under a single market authorization, this is likely to become an unsurmountable burden beyond two or three sites. Unless alternative manufacturing approaches can be found to bridge the regulatory challenge of comparability, realizing a sustainable and investable business model for affordable autologous cell therapy supply is likely to be extremely demanding. Without a proactive approach by the regulators to close this "translational gap," these products may not progress down the development pipeline, threatening patient accessibility to an increasing number of clinician-led autologous cellular therapies that are already demonstrating patient benefits. We propose three prospective manufacturing models for the scale out/roll out of more-than-minimally manipulated clinically led autologous cell therapy products and test their prospects for addressing the challenge of product comparability with a selected expert reference panel of US and UK thought leaders. This paper presents the perspectives and insights of the panel and identifies where operational, technological and scientific improvements should be prioritized. The main purpose of this report is to solicit feedback and seek input from key stakeholders active in the field of autologous cell therapy in establishing a consensus-based manufacturing approach that may permit the roll out of clinically led autologous cell therapies. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Dissimilar Kinetic Behavior of Electrically Manipulated Single- and Double-Stranded DNA Tethered to a Gold Surface

PubMed Central

Rant, Ulrich; Arinaga, Kenji; Tornow, Marc; Kim, Yong Woon; Netz, Roland R.; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard

2006-01-01

We report on the electrical manipulation of single- and double-stranded oligodeoxynucleotides that are end tethered to gold surfaces in electrolyte solution. The response to alternating repulsive and attractive electric surface fields is studied by time-resolved fluorescence measurements, revealing markedly distinct dynamics for the flexible single-stranded and stiff double-stranded DNA, respectively. Hydrodynamic simulations rationalize this finding and disclose two different kinetic mechanisms: stiff polymers undergo rotation around the anchoring pivot point; flexible polymers, on the other hand, are pulled onto the attracting surface segment by segment. PMID:16473909

Dissimilar kinetic behavior of electrically manipulated single- and double-stranded DNA tethered to a gold surface.

PubMed

Rant, Ulrich; Arinaga, Kenji; Tornow, Marc; Kim, Yong Woon; Netz, Roland R; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard

2006-05-15

We report on the electrical manipulation of single- and double-stranded oligodeoxynucleotides that are end tethered to gold surfaces in electrolyte solution. The response to alternating repulsive and attractive electric surface fields is studied by time-resolved fluorescence measurements, revealing markedly distinct dynamics for the flexible single-stranded and stiff double-stranded DNA, respectively. Hydrodynamic simulations rationalize this finding and disclose two different kinetic mechanisms: stiff polymers undergo rotation around the anchoring pivot point; flexible polymers, on the other hand, are pulled onto the attracting surface segment by segment.

Clonogenic assay: adherent cells.

PubMed

Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

2011-03-13

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The effects of parasite age and intensity on variability in acanthocephalan-induced behavioural manipulation.

PubMed

Franceschi, Nathalie; Bauer, Alexandre; Bollache, Loïc; Rigaud, Thierry

2008-08-01

Numerous parasites with complex life cycles are able to manipulate the behaviour of their intermediate host in a way that increases their trophic transmission to the definitive host. Pomphorhynchus laevis, an acanthocephalan parasite, is known to reverse the phototactic behaviour of its amphipod intermediate host, Gammarus pulex, leading to an increased predation by fish hosts. However, levels of behavioural manipulation exhibited by naturally-infected gammarids are extremely variable, with some individuals being strongly manipulated whilst others are almost not affected by infection. To investigate parasite age and parasite intensity as potential sources of this variation, we carried out controlled experimental infections on gammarids using parasites from two different populations. We first determined that parasite intensity increased with exposure dose, but found no relationship between infection and host mortality. Repeated measures confirmed that the parasite alters host behaviour only when it reaches the cystacanth stage which is infective for the definitive host. They also revealed, we believe for the first time, that the older the cystacanth, the more it manipulates its host. The age of the parasite is therefore a major source of variation in parasite manipulation. The number of parasites within a host was also a source of variation. Manipulation was higher in hosts infected by two parasites than in singly infected ones, but above this intensity, manipulation did not increase. Since the development time of the parasite was also different according to parasite intensity (it was longer in doubly infected hosts than in singly infected ones, but did not increase more in multi-infected hosts), individual parasite fitness could depend on the compromise between development time and manipulation efficiency. Finally, the two parasite populations tested induced slightly different degrees of behavioural manipulation.

Control algorithm implementation for a redundant degree of freedom manipulator

NASA Technical Reports Server (NTRS)

Cohan, Steve

1991-01-01

This project's purpose is to develop and implement control algorithms for a kinematically redundant robotic manipulator. The manipulator is being developed concurrently by Odetics Inc., under internal research and development funding. This SBIR contract supports algorithm conception, development, and simulation, as well as software implementation and integration with the manipulator hardware. The Odetics Dexterous Manipulator is a lightweight, high strength, modular manipulator being developed for space and commercial applications. It has seven fully active degrees of freedom, is electrically powered, and is fully operational in 1 G. The manipulator consists of five self-contained modules. These modules join via simple quick-disconnect couplings and self-mating connectors which allow rapid assembly/disassembly for reconfiguration, transport, or servicing. Each joint incorporates a unique drive train design which provides zero backlash operation, is insensitive to wear, and is single fault tolerant to motor or servo amplifier failure. The sensing system is also designed to be single fault tolerant. Although the initial prototype is not space qualified, the design is well-suited to meeting space qualification requirements. The control algorithm design approach is to develop a hierarchical system with well defined access and interfaces at each level. The high level endpoint/configuration control algorithm transforms manipulator endpoint position/orientation commands to joint angle commands, providing task space motion. At the same time, the kinematic redundancy is resolved by controlling the configuration (pose) of the manipulator, using several different optimizing criteria. The center level of the hierarchy servos the joints to their commanded trajectories using both linear feedback and model-based nonlinear control techniques. The lowest control level uses sensed joint torque to close torque servo loops, with the goal of improving the manipulator dynamic behavior. The control algorithms are subjected to a dynamic simulation before implementation.

Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics.

PubMed

Tung, Yi-Chung; Torisawa, Yu-suke; Futai, Nobuyuki; Takayama, Shuichi

2007-11-01

This paper describes a micro flow cytometer system designed for efficient and non-damaging analysis of samples with small numbers of precious cells. The system utilizes actuation of Braille-display pins for micro-scale fluid manipulation and a fluorescence microscope with a CCD camera for optical detection. The microfluidic chip is fully disposable and is composed of a polydimethylsiloxane (PDMS) slab with microchannel features sealed against a thin deformable PDMS membrane. The channels are designed with diffusers to alleviate pulsatile flow behaviors inherent in pin actuator-based peristaltic pumping schemes to maximize hydrodynamic focusing of samples with minimal disturbances in the laminar streams within the channel. A funnel connected to the microfluidic channel is designed for efficient loading of samples with small number of cells and is also positioned on the chip to prevent physical damages of the samples by the squeezing actions of Braille pins during actuation. The sample loading scheme was characterized by both computational fluidic dynamics (CFD) simulation and experimental observation. A fluorescein solution was first used for flow field investigation, followed by use of fluorescence beads with known relative intensities for optical detection performance calibration. Murine myoblast cells (C2C12) were exploited to investigate cell viability for the sample loading scheme of the device. Furthermore, human promyelocytic leukemia (HL60) cells stained by hypotonic DNA staining buffer were also tested in the system for cell cycle analysis. The ability to efficiently analyze cellular samples where the number of cells is small was demonstrated by analyzing cells from a single embryoid body derived from mouse embryonic stem cells. Consequently, the designed microfluidic device reported in this paper is promising for easy-to-use, small sample size flow cytometric analysis, and has potential to be further integrated with other Braille display-based microfluidic devices to facilitate a multi-functional lab-on-a-chip for mammalian cell manipulations.

The radiation hypersensitivity of cells at mitosis.

PubMed

Stobbe, C C; Park, S J; Chapman, J D

2002-12-01

Mitotic cells are hypersensitive to ionizing radiation, exhibiting single-hit inactivation coefficients near to those of repair deficient cell lines and lymphocytes. To elucidate possible mechanisms for this hypersensitivity, the kinetics of oxygen radiosensitization, the proportion of indirect effect by OH radicals and the kinetics of radiation-induced DNA strand breakage in the chromatin of mitotic cells were investigated. Synchronized populations of >90% mitotic HT-29 cells were obtained by the mitotic shake-off method. Cells were irradiated at < or =4 degrees C with (137)Cs gamma-rays. Cellular oxygen concentration was varied by gassing cell suspensions prior to and during irradiation with mixtures of pure N(2) that contained 5% CO(2) and measured quantities of O(2). The indirect effect of OH radicals was investigated with the radical scavenger, DMSO. DNA strand breakage was measured by the comet assay. Mitotic HT-29 cell inactivation is well described by a single-hit inactivation coefficient (alpha) of 1.14 +/- 0.06 Gy(-1). The oxygen enhancement ratio of mitotic cells (at 10% survival) was found to be approximately 2.0, significantly lower than the value of 2.8 measured for interphase (asynchronous) cells. More than 60% of mitotic cell killing was eliminated when the media contained 2 M DMSO, indicating that indirect effect is as important in the killing of mitotic cells as it is for interphase cells. The chromatin in mitotic cells was found to be ~2.8 times more sensitive to radiation-induced DNA single-strand breakage than the chromatin of interphase cells. The alpha-inactivation coefficient of mitotic HT-29 cells was ~30 times larger than that of interphase cells. Mitotic cell chromatin appears to contain intrinsic DNA breaks that are not lethal. In addition, chromatin in mitotic cells was found to be more susceptible to radiation-induced DNA strand-breakage than the dispersed chromatin of interphase cells. How the enhanced production of these simple DNA lesions (that are usually reparable) translates into the lethal (non-reparable) events associated with alpha-inactivation is not known. The compaction/dispersion status of DNA throughout the cell cycle appears to be an important factor for determining intrinsic cell radiosensitivity and might be manipulated for radiotherapeutic advantage.

A portable and integrated instrument for cell manipulation by dielectrophoresis.

PubMed

Burgarella, Sarah; Di Bari, Marco

2015-07-01

The physical manipulation of biological cells is a key point in the development of miniaturized systems for point-of-care analyses. Dielectrophoresis (DEP) has been reported by several laboratories as a promising method in biomedical research for label-free cell manipulation without physical contact, by exploiting the dielectric properties of cells suspended in a microfluidic sample, under the action of high-gradient electric fields. In view of a more extended use of DEP phenomena in lab-on-chip devices for point-of-care settings, we have developed a portable instrument, integrating on the same device the microfluidic biochip for cell manipulation and all the laboratory functions (i.e., DEP electric signal generation, microscopic observation of the biological sample under test and image acquisition) that are normally obtained by combining different nonportable standard laboratory instruments. The nonuniform electric field for cell manipulation on the biochip is generated by microelectrodes, patterned on the silicon substrate of microfluidic channels, using standard microfabrication techniques. Numerical modeling was performed to simulate the electric field distribution, quantify the DEP force, and optimize the geometry of the microelectrodes. The developed instrument includes an electronic board, which allows the control of the electric signal applied to electrodes necessary for DEP, and a miniaturized optical microscope system that allows visual inspection and eventually cell counting, as well as image and video recording. The system also includes the control software. The portable and integrated platform described in this work therefore represents a complete and innovative solution of applied research, suitable for many biological applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An overview of the multi-database manipulation language MDSL

DOE Office of Scientific and Technical Information (OSTI.GOV)

Litwin, W.; Abdellatif, A.

With the increase in availability of databases, data needed by a user are frequently in separate autonomous databases. The logical properties of such data differ from the classical ones with a single database. In particular, they call for new functions for data manipulation. MDSL is a new data manipulation language providing such functions. Most of the MDSL functions are not available in other languages.

Dynamics modeling and adaptive control of flexible manipulators

NASA Technical Reports Server (NTRS)

Sasiadek, J. Z.

1991-01-01

An application of Model Reference Adaptive Control (MRAC) to the position and force control of flexible manipulators and robots is presented. A single-link flexible manipulator is analyzed. The problem was to develop a mathematical model of a flexible robot that is accurate. The objective is to show that the adaptive control works better than 'conventional' systems and is suitable for flexible structure control.

A lightweight, high strength dexterous manipulator for commercial applications

NASA Technical Reports Server (NTRS)

Marzwell, Neville I.; Schena, Bruce M.; Cohan, Steve M.

1991-01-01

The concept, design, and features are described of a lightweight, high strength, modular robot manipulator being developed for space and commercial applications. The manipulator has seven fully active degrees of freedom and is fully operational in 1 G. Each of the seven joints incorporates a unique drivetrain design which provides zero backlash operation, is insensitive to wear, and is single fault tolerant to motor or servo amplifier failure. Feedback sensors provide position, velocity, torque, and motor winding temperature information at each joint. This sensing system is also designed to be single fault tolerant. The manipulator consists of five modules (not including gripper). These modules join via simple quick-disconnect couplings and self-mating connectors which allow rapid assembly and/or disassembly for reconfiguration, transport, or servicing. The manipulator is a completely enclosed assembly, with no exposed components or wires. Although the initial prototype will not be space qualified, the design is well suited to meeting space requirements. The control system provides dexterous motion by controlling the endpoint location and arm pose simultaneously. Potential applications are discussed.

Manipulation of gene expression by infrared laser heat shock and its application to the study of tracheal development in Drosophila.

PubMed

Miao, Guangxia; Hayashi, Shigeo

2015-03-01

Induction of gene expression in a specific cell and a defined time window is desirable to investigate gene function at the cellular level during morphogenesis. To achieve this, we attempted to introduce the infrared laser-evoked gene operator system (IR-LEGO, Kamei et al., 2009) in the Drosophila embryo. In this technique, infrared laser light illumination induces genes to be expressed under the control of heat shock promoters at the single cell level. We applied IR-LEGO to a transgenic fly stock, HS-eGFP, in which the enhanced green fluorescent protein (eGFP) gene is placed under the control of heat shock protein 70 promoter, and showed that eGFP expression can be induced in single cells within 1-2 hr after IR illumination. Furthermore, induction of HS-Branchless transgene encoding the Drosophila fibroblast growth factor (FGF) effectively altered the migration and branching patterns of the tracheal system. Our results indicated that IR-LEGO is a promising choice for the timely control of gene expression in a small group of cells in the Drosophila embryo. By using IR-LEGO, we further demonstrated that the tracheal terminal branching program is sensitive to localized expression of exogenous FGF. © 2014 Wiley Periodicals, Inc.

Estradiol selectively enhances auditory function in avian forebrain neurons

PubMed Central

Caras, Melissa L.; O’Brien, Matthew; Brenowitz, Eliot A.; Rubel, Edwin W

2012-01-01

Sex steroids modulate vertebrate sensory processing, but the impact of circulating hormone levels on forebrain function remains unclear. We tested the hypothesis that circulating sex steroids modulate single-unit responses in the avian telencephalic auditory nucleus, field L. We mimicked breeding or non-breeding conditions by manipulating plasma 17β-estradiol levels in wild-caught female Gambel’s white-crowned sparrows (Zonotrichia leucophrys gambelii). Extracellular responses of single neurons to tones and conspecific songs presented over a range of intensities revealed that estradiol selectively enhanced auditory function in cells that exhibited monotonic rate-level functions to pure tones. In these cells, estradiol treatment increased spontaneous and maximum evoked firing rates, increased pure tone response strengths and sensitivity, and expanded the range of intensities over which conspecific song stimuli elicited significant responses. Estradiol did not significantly alter the sensitivity or dynamic ranges of cells that exhibited non-monotonic rate-level functions. Notably, there was a robust correlation between plasma estradiol concentrations in individual birds and physiological response properties in monotonic, but not non-monotonic neurons. These findings demonstrate that functionally distinct classes of anatomically overlapping forebrain neurons are differentially regulated by sex steroid hormones in a dose-dependent manner. PMID:23223283

[The application of Y-shaped self-expandable covered metal stents in the thoracostomach-airway fistula: a single center, 11 years experience].

PubMed

Fang, Yi; Li, Tengfei; Han, Xinwei; Wu, Gang; Ren, Jianzhuang; Ren, Kewei; Lu, Huibin; Zhang, Quanhui; Li, Zongming

2015-08-01

To investigate the clinical feasibility and efficacy of Y-shaped self-expandable covered metal stents (Y-stents) in the management of thoracostomach-airway fistula. Retrospective analysis was performed for 108 patients treated for thoracostomach-airway fistula with Y-shaped self-expandable coated metal stents between April 2003 and October 2014. Y-stents were designed based on the dimensions of trachea and bronchus and sites of the fistula and then were inserted under DSA monitoring. There were 65 cases with single big Y-stent placement, 26 cases with single small Y-stent placement, 23 cases with double Y-stents placement, and 1 case with 3 Y-stents placement. Stent implantation was successfully accomplished with single manipulation in all patients. Complete occlusion of the fistula was obtained in 104 patients after the primary manipulation, and 4 patients required a secondary manipulation where a double Y-stents was inserted because of failure of primary manipulate. Ninety-two patients completed the follow-up , while 16 were lost. Fifty-nine patients died while 33 were alive with marked improvement in their quality of life. The placement of Y-stents can effectively occlude the thoracostomach-airway fistula in patients who had had the esophageal tumors resected. The technique is not only feasible but reliable to improve the quality of life of the patients.

Manipulating Cells with Static Magnetic Fields

NASA Astrophysics Data System (ADS)

Valles, J. M.; Guevorkian, K.

2005-07-01

We review our investigations of the use of static magnetic fields, B, for manipulating cells and cellular processes. We describe how B fields modify the cell division pattern of frog embryos and consequently can be used to probe the pattern determinants. We also observe that magnetic fields modify the swimming behavior of Paramecium Caudatum. We describe these modifications and their potential application to investigations of their swimming behavior.

Rapid and continuous magnetic separation in droplet microfluidic devices.

PubMed

Brouzes, Eric; Kruse, Travis; Kimmerling, Robert; Strey, Helmut H

2015-02-07

We present a droplet microfluidic method to extract molecules of interest from a droplet in a rapid and continuous fashion. We accomplish this by first marginalizing functionalized super-paramagnetic beads within the droplet using a magnetic field, and then splitting the droplet into one droplet containing the majority of magnetic beads and one droplet containing the minority fraction. We quantitatively analysed the factors which affect the efficiency of marginalization and droplet splitting to optimize the enrichment of magnetic beads. We first characterized the interplay between the droplet velocity and the strength of the magnetic field and its effect on marginalization. We found that marginalization is optimal at the midline of the magnet and that marginalization is a good predictor of bead enrichment through splitting at low to moderate droplet velocities. Finally, we focused our efforts on manipulating the splitting profile to improve the enrichment provided by asymmetric splitting. We designed asymmetric splitting forks that employ capillary effects to preferentially extract the bead-rich regions of the droplets. Our strategy represents a framework to optimize magnetic bead enrichment methods tailored to the requirements of specific droplet-based applications. We anticipate that our separation technology is well suited for applications in single-cell genomics and proteomics. In particular, our method could be used to separate mRNA bound to poly-dT functionalized magnetic microparticles from single cell lysates to prepare single-cell cDNA libraries.

Rapid and continuous magnetic separation in droplet microfluidic devices

PubMed Central

Brouzes, Eric; Kruse, Travis; Kimmerling, Robert; Strey, Helmut H.

2015-01-01

We present a droplet microfluidic method to extract molecules of interest from a droplet in a rapid and continuous fashion. We accomplish this by first marginalizing functionalized super-paramagnetic beads within the droplet using a magnetic field, and then splitting the droplet into one droplet containing the majority of magnetic beads and one droplet containing the minority fraction. We quantitatively analysed the factors which affect the efficiency of marginalization and droplet splitting to optimize the enrichment of magnetic beads. We first characterized the interplay between the droplet velocity and the strength of the magnetic field and its effect on marginalization. We found that marginalization is optimal at the midline of the magnet and that marginalization is a good predictor of bead enrichment through splitting at low to moderate droplet velocities. Finally, we focused our efforts on manipulating the splitting profile to improve the enrichment provided by asymmetric splitting. We designed asymmetric splitting forks that employ capillary effects to preferentially extract the bead-rich regions of the droplets. Our strategy represents a framework to optimize magnetic bead enrichment methods tailored to the requirements of specific droplet-based applications. We anticipate that our separation technology is well suited for applications in single-cell genomics and proteomics. In particular, our method could be used to separate mRNA bound to poly-dT functionalized magnetic microparticles from single cell lysates to prepare single-cell cDNA libraries. PMID:25501881

Micromagnet arrays for on-chip focusing, switching, and separation of superparamagnetic beads and single cells.

PubMed

Rampini, S; Kilinc, D; Li, P; Monteil, C; Gandhi, D; Lee, G U

2015-08-21

Nonlinear magnetophoresis (NLM) is a novel approach for on-chip transport and separation of superparamagnetic (SPM) beads, based on a travelling magnetic field wave generated by the combination of a micromagnet array (MMA) and an applied rotating magnetic field. Here, we present two novel MMA designs that allow SPM beads to be focused, sorted, and separated on-chip. Converging MMAs were used to rapidly collect the SPM beads from a large region of the chip and focus them into synchronised lines. We characterise the collection efficiency of the devices and demonstrate that they can facilitate on-chip analysis of populations of SPM beads using a single-point optical detector. The diverging MMAs were used to control the transport of the beads and to separate them based on their size. The separation efficiency of these devices was determined by the orientation of the magnetisation of the micromagnets relative to the external magnetic field and the size of the beads and relative to that of micromagnets. By controlling these parameters and the rotation of the external magnetic field we demonstrated the controlled transport of SPM bead-labelled single MDA-MB-231 cells. The use of these novel MMAs promises to allow magnetically-labelled cells to be efficiently isolated and then manipulated on-chip for analysis with high-resolution chemical and physical techniques.

Clonal expansion under the microscope: studying lymphocyte activation and differentiation using live-cell imaging.

PubMed

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.

Transgenic manipulation of the metabolism of polyamines in poplar cells

Treesearch

Pratiksha Bhatnagar; Bernadette M. Glasheen; Suneet K. Bains; Stephanie L. Long; Rakesh Minocha; Christian Walter; Subhash C. Minocha

2001-01-01

The metabolism of polyamines (putrescine, spermidine, and spermine) has become the target of genetic manipulation because of their significance in plant development and possibly stress tolerance. We studied the polyamine metabolism in non-transgenic (NT) and transgenic cells of poplar (Populus nigra 3 maximowiczii) expressing a...

DNA mechanics as a tool to probe helicase and translocase activity.

PubMed

Lionnet, Timothée; Dawid, Alexandre; Bigot, Sarah; Barre, François-Xavier; Saleh, Omar A; Heslot, François; Allemand, Jean-François; Bensimon, David; Croquette, Vincent

2006-01-01

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.

Lab-on-a-chip Single Particle Dielectrophoretic Traps

NASA Astrophysics Data System (ADS)

Wang, Weina; Shao, Hua; Lear, Kevin

2007-03-01

Cell-patterning and cell-manipulation in micro-environments are fundamental to biological and biomedical applications, for example, spectroscopic cytology based cancer detection. Dielectrophoresis (DEP) traps with transparent centers for stabilized cell and particle optofluidic intracavity spectroscopy (OFIS) were fabricated by patterning 10 μm wide, planar gold electrodes on glass substrates. The capturing strength of DEP traps was quantified based on the minimum AC voltage required to capture and hold varying diameter polystyrene or was it some other material, e.g. silica or PMMA microspheres in water as a function of frequency required under a constant flowrate of 20 μm/s. The maximum required trapping voltage in the negative DEP regime of f = 1 kHz to 40 MHz was 5.0 VAC. The use of AC fields effectively suppressed hydrolysis. New geometries of DEP traps are being explored on the basis of 3-D electrostatic field simulations.

Scaling of membrane-type locally resonant acoustic metamaterial arrays.

PubMed

Naify, Christina J; Chang, Chia-Ming; McKnight, Geoffrey; Nutt, Steven R

2012-10-01

Metamaterials have emerged as promising solutions for manipulation of sound waves in a variety of applications. Locally resonant acoustic materials (LRAM) decrease sound transmission by 500% over acoustic mass law predictions at peak transmission loss (TL) frequencies with minimal added mass, making them appealing for weight-critical applications such as aerospace structures. In this study, potential issues associated with scale-up of the structure are addressed. TL of single-celled and multi-celled LRAM was measured using an impedance tube setup with systematic variation in geometric parameters to understand the effects of each parameter on acoustic response. Finite element analysis was performed to predict TL as a function of frequency for structures with varying complexity, including stacked structures and multi-celled arrays. Dynamic response of the array structures under discrete frequency excitation was investigated using laser vibrometry to verify negative dynamic mass behavior.

Formalin-inactivated Venezuelan Equine Encephalomyelitis (Trinidad Strain) Vaccine Produced in Rolling-Bottle Cultures of Chicken Embryo Cells

PubMed Central

Cole, Francis E.; May, Stephen W.; Robinson, David M.

1973-01-01

Formalin-inactivated Venezuelan equine encephalomyelitis vaccine was prepared from virus grown in rolling-bottle cultures of chicken embryo cells. Trinidad strain virus was propagated in these cultures with a maintenance medium consisting of serum-free medium 199 containing 0.25% human serum albumin (USP) and antibiotics. Manipulation of multiplicity of inoculum (0.06 to 0.00006) and maintenance medium volume (100 to 300 ml) resulted in high-titered virus yields and only moderate cell destruction when fluids from infected cultures were harvested at 18 to 24 hr. The virus was inactivated at 37 C by 0.05% Formalin within 8 to 10 hr and with 0.1% Formalin within 6 to 8 hr. Single dose, antigen extinction tests in mice performed with 30 small-scale vaccine lots showed excellent potency at either Formalin concentration with inactivation periods ranging from 24 to 96 hr. PMID:4694345

Hyperchromatic laser scanning cytometry

NASA Astrophysics Data System (ADS)

Tárnok, Attila; Mittag, Anja

2007-02-01

In the emerging fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics multi- and polychromatic analysis of biological specimens has become increasingly important. Combining different technologies and staining methods polychromatic analysis (i.e. using 8 or more fluorescent colors at a time) can be pushed forward to measure anything stainable in a cell, an approach termed hyperchromatic cytometry. For cytometric cell analysis microscope based Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide. Based on the feature of relocation identical cells can be subsequently reanalyzed. In this manner data on the single cell level after manipulation steps can be collected. In this overview various components for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA): 1) polychromatic cytometry, 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). With the intelligent combination of several of these techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The combination of high-throughput and high-content SBC analysis with high-resolution confocal imaging allows clear verification of phenotypically distinct subpopulations of cells with structural information. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

Extracellular calcium controls the expression of two different forms of ripple-like hippocampal oscillations.

PubMed

Aivar, Paloma; Valero, Manuel; Bellistri, Elisa; Menendez de la Prida, Liset

2014-02-19

Hippocampal high-frequency oscillations (HFOs) are prominent in physiological and pathological conditions. During physiological ripples (100-200 Hz), few pyramidal cells fire together coordinated by rhythmic inhibitory potentials. In the epileptic hippocampus, fast ripples (>200 Hz) reflect population spikes (PSs) from clusters of bursting cells, but HFOs in the ripple and the fast ripple range are vastly intermixed. What is the meaning of this frequency range? What determines the expression of different HFOs? Here, we used different concentrations of Ca(2+) in a physiological range (1-3 mM) to record local field potentials and single cells in hippocampal slices from normal rats. Surprisingly, we found that this sole manipulation results in the emergence of two forms of HFOs reminiscent of ripples and fast ripples recorded in vivo from normal and epileptic rats, respectively. We scrutinized the cellular correlates and mechanisms underlying the emergence of these two forms of HFOs by combining multisite, single-cell and paired-cell recordings in slices prepared from a rat reporter line that facilitates identification of GABAergic cells. We found a major effect of extracellular Ca(2+) in modulating intrinsic excitability and disynaptic inhibition, two critical factors shaping network dynamics. Moreover, locally modulating the extracellular Ca(2+) concentration in an in vivo environment had a similar effect on disynaptic inhibition, pyramidal cell excitability, and ripple dynamics. Therefore, the HFO frequency band reflects a range of firing dynamics of hippocampal networks.

Imaging and manipulation of adatoms on an alumina surface by noncontact atomic force microscopy

NASA Astrophysics Data System (ADS)

Simon, G. H.; Heyde, M.; Freund, H.-J.

2012-02-01

Noncontact atomic force microscopy (NC-AFM) has been performed on an aluminum oxide film grown on NiAl(110) in ultrahigh vacuum (UHV) at low temperature (5 K). Results reproduce the topography of the structural model, unlike scanning tunnelling microscopy (STM) images. Equipped with this extraordinary contrast the network of extended defects, which stems from domain boundaries intersecting the film surface, can be analysed in atomic detail. The knowledge of occurring surface structures opens up the opportunity to determine adsorption sites of individual adsorbates on the alumina film. The level of difficulty for such imaging depends on the imaging characteristics of the substrate and the interaction which can be maintained above the adsorbate. Positions of single adsorbed gold atoms within the unit cell have been determined despite their easy removal at slightly higher interaction strength. Preliminary manipulation experiments indicate a pick-up process for the vanishing of the gold adatoms from the film surface.

Cell Internal Treatable Microplasma Jets in Cancer Therapies

NASA Astrophysics Data System (ADS)

Kim, Jae Young; Wei, Yanzhang; Li, Jinhua; Kim, Sung-O.

2011-10-01

We developed a 15- μm-sized, single-cellular-level, and cell-manipulatable microplasma jet device with a microcapillary glass tip and described its potential in physical cancer therapies. The microcapillary tip is a funnel shaped glass tube and its nozzle has an inner diameter of 15 μm and an outer diameter of 20 μm with 20 capillary angle. The electrical and optical properties of this plasma jet and apoptosis results of cultured murine B16F0 melanoma tumor cells and CL.7 fibroblast cells treated with the plasma jets were described. In spite of the small inner diameter and the low gas flow rate of the microplasma jet device, the generated plasma jets are stable enough to treat tumor cells. The microplasma jet was observed inducing apoptosis in cultured murine melanoma tumor cells in a dose-dependent manner. Furthermore, the percentage of apoptotic cells of murine melanoma tumor cells induced by this plasma device was approximately 2.5 times bigger than that of murine fibroblast cells as indicated by an Annex V-FITC method. This highly precise plasma medicine, which enables new directed cancer therapies, can be combined with current cell manipulation and cell culturing technologies without much difficulty.

Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells

PubMed Central

Kornete, Mara

2018-01-01

Adoptive cell transfer is an important approach for basic research and emerges as an effective treatment for various diseases, including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. In this article, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms in up to 25% of the transfected primary T cells. We used gene editing–mediated allele switching to quantify homology-directed repair, systematically optimize experimental parameters, and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells, with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Repairing the cause of the scurfy syndrome, a 2-bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells. PMID:29445007

Phospholipase C mediated Suppression of Dark Noise Enables Single Photon Detection in Drosophila Photoreceptors

PubMed Central

Katz, Ben; Minke, Baruch

2012-01-01

Drosophila photoreceptor cells use the ubiquitous G-protein-mediated phospholipase C (PLC) cascade to achieve ultimate single photon sensitivity. This is manifested in the single photon responses (quantum bumps). In photoreceptor cells, dark activation of Gqα molecules occurs spontaneously and produces unitary dark events (dark bumps). A high rate of spontaneous Gqα activation and dark bump production potentially hampers single photon detection. We found that in wild type flies the in vivo rate of spontaneous Gqα activation is very high. Nevertheless, this high rate is not manifested in a substantially high rate of dark bumps. Therefore, it is unclear how phototransduction suppresses dark bump production, arising from spontaneous Gqα activation, while still maintaining high-fidelity representation of single photons. In this study we show that reduced PLC catalytic activity selectively suppressed production of dark bumps but not light-induced bumps. Manipulations of PLC activity using PLC mutant flies and Ca2+ modulations revealed that a critical level of PLC activity is required to induce bump production. The required minimal level of PLC activity, selectively suppressed random production of single Gqα-activated dark bumps despite a high rate of spontaneous Gqα activation. This minimal PLC activity level is reliably obtained by photon induced synchronized activation of several neighboring Gqα molecules activating several PLC molecules, but not by random activation of single Gqα molecules. We thus demonstrate how a G-protein-mediated transduction system, with PLC as its target, selectively suppresses its intrinsic noise while preserving reliable signaling. PMID:22357856

Manipulation of micro-objects using acoustically oscillating bubbles based on the gas permeability of PDMS.

PubMed

Liu, Bendong; Tian, Baohua; Yang, Xu; Li, Mohan; Yang, Jiahui; Li, Desheng; Oh, Kwang W

2018-05-01

This paper presents a novel manipulation method for micro-objects using acoustically oscillating bubbles with a controllable position based on the gas permeability of polydimethylsiloxane. The oscillating bubble trapped within the side channel attracts the neighboring micro-objects, and the position of the air-liquid interface is controlled by generating temporary pressure difference between the side channel and the air channel. To demonstrate the feasibility of the method in technological applications, polystyrene microparticles of 10  μ m in diameter were successfully captured, transported, and released. The influence of pressure difference on the movement speed of the air-liquid interface was demonstrated in our experiments, and the manipulation performance was also characterized by varying the frequency of the acoustic excitation and the pressure difference. Since the bubble generation and the air-liquid interface movement in our manipulation method do not need any electrochemical reaction and any high temperature, this on-chip manipulation method provides a controllable, efficient, and noninvasive tool for handling micro-objects such as particles, cells, and other entities. The whole manipulation process, including capturing, transporting, and releasing of particles, spent less than 1 min. It can be used to select the cells and particles in the microfluidic device or change the cell culture medium.

Three-dimensional visualization and a deep-learning model reveal complex fungal parasite networks in behaviorally manipulated ants

PubMed Central

Fredericksen, Maridel A.; Zhang, Yizhe; Hazen, Missy L.; Loreto, Raquel G.; Mangold, Colleen A.; Chen, Danny Z.; Hughes, David P.

2017-01-01

Some microbes possess the ability to adaptively manipulate host behavior. To better understand how such microbial parasites control animal behavior, we examine the cell-level interactions between the species-specific fungal parasite Ophiocordyceps unilateralis sensu lato and its carpenter ant host (Camponotus castaneus) at a crucial moment in the parasite’s lifecycle: when the manipulated host fixes itself permanently to a substrate by its mandibles. The fungus is known to secrete tissue-specific metabolites and cause changes in host gene expression as well as atrophy in the mandible muscles of its ant host, but it is unknown how the fungus coordinates these effects to manipulate its host’s behavior. In this study, we combine techniques in serial block-face scanning-electron microscopy and deep-learning–based image segmentation algorithms to visualize the distribution, abundance, and interactions of this fungus inside the body of its manipulated host. Fungal cells were found throughout the host body but not in the brain, implying that behavioral control of the animal body by this microbe occurs peripherally. Additionally, fungal cells invaded host muscle fibers and joined together to form networks that encircled the muscles. These networks may represent a collective foraging behavior of this parasite, which may in turn facilitate host manipulation. PMID:29114054

Moving messages in the developing brain—emerging roles for mRNA transport and local translation in neural stem cells

PubMed Central

Pilaz, Louis-Jan; Silver, Debra L.

2017-01-01

The mammalian cerebral cortex is a complex brain structure integral to our higher cognition. During embryonic cortical development, radial glial progenitors (RGCs) produce neurons and serve as physical structures for migrating neurons. Recent discoveries highlight new roles for RNA localization and local translation in RGCs, both at the cell body and at distal structures called basal endfeet. By implementing technologies from the field of RNA research to brain development, investigators can manipulate RNA-binding proteins as well as visualize single-molecule RNAs, live movement of mRNAs and their binding proteins, and translation. Going forward, these studies establish a framework for investigating how post-transcriptional RNA regulation helps shape RGC function and triggers neurodevelopmental diseases. PMID:28304078

Micro-magnetic Structures for Biological Applications

NASA Astrophysics Data System (ADS)

Howdyshell, Marci L.

Developments in single-molecule and single-cell experiments over the past century have provided researchers with many tools to probe the responses of cells to stresses such as physical force or to the injection of foreign genes. Often these techniques target the cell membrane, although many are now advancing to probe within the cell. As these techniques are improved upon and the investigations advance toward clinical applications, it has become more critical to achieve high-throughput outcomes which in turn lead to statistically significant results. The technologies developed in this thesis are targeted at transfecting large populations of cells with controlled doses of specific exogenic material without adversely affecting cell viability. Underlying this effort is a platform of lithographically patterned ferromagnetic thin films capable of remotely manipulating and localizing magnetic microbeads attached to biological entities. A novel feature of this approach, as demonstrated here with both DNA and cells, is the opportunity for multiplexed operations on targeted biological specimens. This thesis includes two main thrusts: (1) the advancement of the trapping platforms through experimental verification of mathematical models providing the energy landscapes associated with the traps and (2) implementation of the platform as a basis for rapid and effective high-throughput microchannel and nanochannel cell electroporation devices. The electroporation devices have, in our studies, not only been demonstrated to sustain cell viability with extremely low cell mortality rates, but are also found to be effective for various types of cells. The advances over current electroporation technologies that are achieved in these efforts demonstrate the potential for detection of mRNA expression in heterogeneous cell populations and probing intracellular responses to the introduction of foreign genes into cells.

Clinical use of adipose-derived stem cells: European legislative issues.

PubMed

Raposio, Edoardo; Ciliberti, RosaGemma

2017-12-01

With this study we analyse the current European legislation in order to provide guidance for regenerative medicine professionals on correct Adipose-derived Stem Cells (ASCs) isolation and use protocols for clinical applications. The European Medicines Agency (EMA) considers that ASCs does not fall within the definition of an advanced therapy medicinal product if the cells have not been subjected to a substantial manipulation, and the mode of action of the cells (contribute to and enhance tissue renewal and turnover of the subcutaneous tissue) is considered to be homologous to the donor fat tissue. Collagenase digestion, as well as cell culturing, is considered to be a substantial manipulation. Only transplantation of a non-manipulated tissue to another location in the same anatomical or histological environment is considered to be homologous. According to these considerations, ASCs should be not-cultured, isolated mechanically and used only in the subcutaneous tissue.

Not Just a Sum? Identifying Different Types of Interplay between Constituents in Combined Interventions

PubMed Central

Van Deun, Katrijn; Thorrez, Lieven; van den Berg, Robert A.; Smilde, Age K.; Van Mechelen, Iven

2015-01-01

Motivation Experiments in which the effect of combined manipulations is compared with the effects of their pure constituents have received a great deal of attention. Examples include the study of combination therapies and the comparison of double and single knockout model organisms. Often the effect of the combined manipulation is not a mere addition of the effects of its constituents, with quite different forms of interplay between the constituents being possible. Yet, a well-formalized taxonomy of possible forms of interplay is lacking, let alone a statistical methodology to test for their presence in empirical data. Results Starting from a taxonomy of a broad range of forms of interplay between constituents of a combined manipulation, we propose a sound statistical hypothesis testing framework to test for the presence of each particular form of interplay. We illustrate the framework with analyses of public gene expression data on the combined treatment of dendritic cells with curdlan and GM-CSF and show that these lead to valuable insights into the mode of action of the constituent treatments and their combination. Availability and Implementation R code implementing the statistical testing procedure for microarray gene expression data is available as supplementary material. The data are available from the Gene Expression Omnibus with accession number GSE32986. PMID:25965065

Not Just a Sum? Identifying Different Types of Interplay between Constituents in Combined Interventions.

PubMed

Van Deun, Katrijn; Thorrez, Lieven; van den Berg, Robert A; Smilde, Age K; Van Mechelen, Iven

2015-01-01

Experiments in which the effect of combined manipulations is compared with the effects of their pure constituents have received a great deal of attention. Examples include the study of combination therapies and the comparison of double and single knockout model organisms. Often the effect of the combined manipulation is not a mere addition of the effects of its constituents, with quite different forms of interplay between the constituents being possible. Yet, a well-formalized taxonomy of possible forms of interplay is lacking, let alone a statistical methodology to test for their presence in empirical data. Starting from a taxonomy of a broad range of forms of interplay between constituents of a combined manipulation, we propose a sound statistical hypothesis testing framework to test for the presence of each particular form of interplay. We illustrate the framework with analyses of public gene expression data on the combined treatment of dendritic cells with curdlan and GM-CSF and show that these lead to valuable insights into the mode of action of the constituent treatments and their combination. R code implementing the statistical testing procedure for microarray gene expression data is available as supplementary material. The data are available from the Gene Expression Omnibus with accession number GSE32986.

Manipulating the content of dynamic natural scenes to characterize response in human MT/MST.

PubMed

Durant, Szonya; Wall, Matthew B; Zanker, Johannes M

2011-09-09

Optic flow is one of the most important sources of information for enabling human navigation through the world. A striking finding from single-cell studies in monkeys is the rapid saturation of response of MT/MST areas with the density of optic flow type motion information. These results are reflected psychophysically in human perception in the saturation of motion aftereffects. We began by comparing responses to natural optic flow scenes in human visual brain areas to responses to the same scenes with inverted contrast (photo negative). This changes scene familiarity while preserving local motion signals. This manipulation had no effect; however, the response was only correlated with the density of local motion (calculated by a motion correlation model) in V1, not in MT/MST. To further investigate this, we manipulated the visible proportion of natural dynamic scenes and found that areas MT and MST did not increase in response over a 16-fold increase in the amount of information presented, i.e., response had saturated. This makes sense in light of the sparseness of motion information in natural scenes, suggesting that the human brain is well adapted to exploit a small amount of dynamic signal and extract information important for survival.

Letters: Comment on "Effects of multi-scattering on the performance of a single-beam acoustic manipulation device".

PubMed

Mitri, F G

2013-11-01

The concern addressed in the present commentary is to point out the omission of the azimuthal component Fφ of the axial acoustic radiation force provided in M. Azarpeyvand, M. A. Alibakhshi, R. Self, "Effects of multi-scattering on the performance of a single-beam acoustic manipulation device," IEEE Trans. Ultrason. Ferroelectr. Freq. Control, vol. 59, no. 8, pp. 1741-1749, 2012, which may suggest a miscalculation of the radiation force function Ym and its related numerical computations.

Genetic modification of stem cells for improved therapy of the infarcted myocardium.

PubMed

Haider, Husnain Kh; Mustafa, Anique; Feng, Yuliang; Ashraf, Muhammad

2011-10-03

The conventional treatment modalities for ischemic heart disease only provide symptomatic relief to the patient without repairing and regenerating the damaged myocardium. Stem cell transplantation has emerged as a promising alternative therapeutic approach for cardiovascular diseases. Stem cells possess the potential of differentiation to adopt morphofunctional cardiac and vasculogenic phenotypes to repopulate the scar tissue and restore regional blood flow in the ischemic myocardium. These beneficial therapeutic effects make stem cell transplantation the method of choice for the treatment of ischemic heart disease. The efficacy of stem cell transplantation may be augmented by genetic manipulation of the cells prior to transplantation. Not only will insertion of therapeutic transgene(s) into the stem cells support the survival and differentiation of cells in the unfavorable microenvironment of the ischemic myocardium, but also the genetically manipulated stem cells will serve as a source of the transgene expression product in the heart for therapeutic benefits. We provide an overview of the extensively studied stem cell types for cardiac regeneration, the various methods in which these cells have been genetically manipulated and rationale of genetic modification of stem cells for use in regenerative cardiovascular therapeutics.

Dual stimuli-responsive smart beads that allow "on-off" manipulation of cancer cells.

PubMed

Kim, Young-Jin; Kim, Soo Hyeon; Fujii, Teruo; Matsunaga, Yukiko T

2016-06-24

Temperature- and electric field-responsive polymer-conjugated polystyrene beads, termed smart beads, are designed to isolate cancer cells. In smart beads, the reversible "on-off" antigen-antibody reaction and dielectrophoresis force on an electrode are accomplished to realize "on-off" remote manipulation of smart beads and cancer cells. Both the zeta-potential and the hydrodynamic diameter of the smart beads are sensitive to temperature, allowing "on-off" reversible capture and release of cancer cells. Cancer cell-captured smart beads are then localized on electrodes by applying an electrical signal.

Actin Cytoskeleton Manipulation by Effector Proteins Secreted by Diarrheagenic Escherichia coli Pathotypes

PubMed Central

Navarro-Garcia, Fernando; Serapio-Palacios, Antonio; Ugalde-Silva, Paul; Tapia-Pastrana, Gabriela; Chavez-Dueñas, Lucia

2013-01-01

The actin cytoskeleton is a dynamic structure necessary for cell and tissue organization, including the maintenance of epithelial barriers. Disruption of the epithelial barrier coincides with alterations of the actin cytoskeleton in several disease states. These disruptions primarily affect the paracellular space, which is normally regulated by tight junctions. Thereby, the actin cytoskeleton is a common and recurring target of bacterial virulence factors. In order to manipulate the actin cytoskeleton, bacteria secrete and inject toxins and effectors to hijack the host cell machinery, which interferes with host-cell pathways and with a number of actin binding proteins. An interesting model to study actin manipulation by bacterial effectors is Escherichia coli since due to its genome plasticity it has acquired diverse genetic mobile elements, which allow having different E. coli varieties in one bacterial species. These E. coli pathotypes, including intracellular and extracellular bacteria, interact with epithelial cells, and their interactions depend on a specific combination of virulence factors. In this paper we focus on E. coli effectors that mimic host cell proteins to manipulate the actin cytoskeleton. The study of bacterial effector-cytoskeleton interaction will contribute not only to the comprehension of the molecular causes of infectious diseases but also to increase our knowledge of cell biology. PMID:23509714

An integrated centrifugo-opto-microfluidic platform for arraying, analysis, identification and manipulation of individual cells.

PubMed

Burger, R; Kurzbuch, D; Gorkin, R; Kijanka, G; Glynn, M; McDonagh, C; Ducrée, J

2015-01-21

In this work we present a centrifugal microfluidic system enabling highly efficient collective trapping and alignment of particles such as microbeads and cells, their multi-colour fluorescent detection and subsequent manipulation by optical tweezers. We demonstrate array-based capture and imaging followed by "cherry-picking" of individual particles, first for fluorescently labelled polystyrene (PS) beads and then for cells. Different cell lines are discriminated based on intracellular as well as surface-based markers.

Kinematics modeling and experimentation of the multi-manipulator tooth-arrangement robot for full denture manufacturing.

PubMed

Zhang, Yong-de; Jiang, Jin-gang; Liang, Ting; Hu, Wei-ping

2011-12-01

Artificial teeth are very complicated in shape, and not easy to be grasped and manipulated accurately by a single robot. The method of tooth-arrangement by multi-manipulator for complete denture manufacturing proposed in this paper. A novel complete denture manufacturing mechanism is designed based on multi-manipulator and dental arch generator. Kinematics model of the multi-manipulator tooth-arrangement robot is built by analytical method based on tooth-arrangement principle for full denture. Preliminary experiments on tooth-arrangement are performed using the multi-manipulator tooth-arrangement robot prototype system. The multi-manipulator tooth-arrangement robot prototype system can automatically design and manufacture a set of complete denture that is suitable for a patient according to the jaw arch parameters. The experimental results verified the validity of kinematics model of the multi-manipulator tooth-arrangement robot and the feasibility of the manufacture strategy of complete denture fulfilled by multi-manipulator tooth-arrangement robot.

Kinematic Determination of an Unmodeled Serial Manipulator by Means of an IMU

NASA Astrophysics Data System (ADS)

Ciarleglio, Constance A.

Kinematic determination for an unmodeled manipulator is usually done through a-priori knowledge of the manipulator physical characteristics or external sensor information. The mathematics of the kinematic estimation, often based on Denavit- Hartenberg convention, are complex and have high computation requirements, in addition to being unique to the manipulator for which the method is developed. Analytical methods that can compute kinematics on-the fly have the potential to be highly beneficial in dynamic environments where different configurations and variable manipulator types are often required. This thesis derives a new screw theory based method of kinematic determination, using a single inertial measurement unit (IMU), for use with any serial, revolute manipulator. The method allows the expansion of reconfigurable manipulator design and simplifies the kinematic process for existing manipulators. A simulation is presented where the theory of the method is verified and characterized with error. The method is then implemented on an existing manipulator as a verification of functionality.

Wireless-powered electroactive soft microgripper

NASA Astrophysics Data System (ADS)

Cheong, Hau Ran; Teo, Choon Yee; Leow, Pei Ling; Lai, Koon Chun; Chee, Pei Song

2018-05-01

This paper presents a wireless powered single active finger ionic polymer metal composite (IPMC) based microgripper that is operated using external radio-frequency (RF) magnetic field for biological cell manipulation application. A unimorph-like active finger is fabricated by integrating the IPMC actuator to the planar resonant LC receiver and DC rectifier circuits (made of flexible double-sided copper clad polyimide). The finger activated when the device is exposed to the external magnetic field generated by transmitter circuit that matches the resonant frequency of LC receiver circuit, ∼13.6 MHz in magnetic resonant coupling power transfer mechanism. The fabricated prototype shows a maximum IPMC deflection of 0.765 mm (activation force of 0.17 mN) at the RF power of 0.65 W with 3.5 VDC supplied from the LC receiver circuit. Three repeated ON-OFF wireless activation cycle was performed with the reported cumulative deflection of 0.57 mm. The cumulative deflection was increased to 1.17 mm, 1.19 mm and 1.24 mm for three different samples respectively at 5 VDC supplied. As a proof of concept, fish egg was used to represent the biological cell manipulation operation. The microgripper successfully gripped the fish egg sample without any damages. The experiments result validates the effectiveness of wireless RF soft microgripper towards the target application.

Acoustofluidic waveguides for localized control of acoustic wavefront in microfluidics

PubMed Central

Bian, Yusheng; Guo, Feng; Yang, Shujie; Mao, Zhangming; Bachman, Hunter; Tang, Shi-Yang; Ren, Liqiang; Zhang, Bin; Gong, Jianying; Guo, Xiasheng

2017-01-01

The precise manipulation of acoustic fields in microfluidics is of critical importance for the realization of many biomedical applications. Despite the tremendous efforts devoted to the field of acoustofluidics during recent years, dexterous control, with an arbitrary and complex acoustic wavefront, in a prescribed, microscale region is still out of reach. Here, we introduce the concept of acoustofluidic waveguide, a three-dimensional compact configuration that is capable of locally guiding acoustic waves into a fluidic environment. Through comprehensive numerical simulations, we revealed the possibility of forming complex field patterns with defined pressure nodes within a highly localized, pre-determined region inside the microfluidic chamber. We also demonstrated the tunability of the acoustic field profile through controlling the size and shape of the waveguide geometry, as well as the operational frequency of the acoustic wave. The feasibility of the waveguide concept was experimentally verified via microparticle trapping and patterning. Our acoustofluidic waveguiding structures can be readily integrated with other microfluidic configurations and can be further designed into more complex types of passive acoustofluidic devices. The waveguide platform provides a promising alternative to current acoustic manipulation techniques and is useful in many applications such as single-cell analysis, point-of-care diagnostics, and studies of cell–cell interactions. PMID:29358901

[Are there alternative forms of therapy in breast carcinoma? Status and perspectives for the treatment of metastasized breast carcinoma].

PubMed

Unger, C; Marmé, D

1995-03-28

The emergence of new cytotoxic agents and techniques for treatment of systemic disease as single modalities or in combination with irradiation and surgery will impact on the use of such agents in the management of systemic breast cancer. Metastatic breast carcinoma, unlike other solid tumors, is highly responsive to chemotherapy, response rates of 50 to 70% have been reported consistently, although there has not been a significant improvement on long-term survival of these patients in the last ten years. New therapeutic approaches include cytotoxic and hormonal agents, growth and differentiation factors, monoclonal antibodies, hematopoietic stem cell support, conquest of tumor cell resistance by MDR-modulation, genetic manipulation, identification of new targets on the tumor surface, synthesis of target-oriented designer-drugs and inhibition of tumor angiogenesis. In breast cancer the tumor growth correlates with vascularization and angiogenesis. Tumor angiogenesis is stimulated by the vascular endothelial growth factor (VEGF). Microvessel density is a significant predictor of survival among node-negative women, who are at risk for having occult metastases at presentation. These patients could then be given systemic adjuvant therapy. Animal experiments show promising inhibition of tumor growth in nude mice after application of antibodies against VEGF. Other methods of manipulation of molecular mechanisms of angiogenesis are under investigation.

A Prospective, Randomized, Masked, and Placebo-Controlled Efficacy Study of Intraarticular Allogeneic Adipose Stem Cells for the Treatment of Osteoarthritis in Dogs

PubMed Central

Harman, Robert; Carlson, Kim; Gaynor, Jamie; Gustafson, Scott; Dhupa, Sarit; Clement, Keith; Hoelzler, Michael; McCarthy, Tim; Schwartz, Pamela; Adams, Cheryl

2016-01-01

Osteoarthritis (OA) is a degenerative joint disease with a high prevalence in dogs. Mesenchymal stem cells (MSCs) have been used to treat humans, dogs, and horses with OA. This report describes a prospective, randomized, blinded, and placebo-controlled clinical efficacy study of intraarticular allogeneic adipose stem cells for the treatment of dogs with OA. Health assessments and measurements of pain and activity impairment were performed at baseline and at selected time points through day 60. The primary outcome variable was the owner Client-Specific Outcome Measurement (CSOM) and secondary measures included veterinary pain on manipulation, veterinary global score, and owner global score. The dogs were treated with either a saline placebo or a single dose of allogeneic adipose-derived MSCs in either one or two joints. Seventy-four dogs were statistically analyzed for efficacy outcomes. Success in the primary outcome variable, CSOM, was statistically improved in the treated dogs compared to the placebo dogs (79.2 versus 55.4%, p = 0.029). The veterinary pain on manipulation score (92.8 versus 50.2%, p = 0.017) and the veterinary global score (86.9 versus 30.8%, p = 0.009) were both statistically improved in treated dogs compared to placebo. There was no detected significant difference between treated and placebo dogs in the incidence of adverse events or negative health findings. Allogeneic adipose-derived stem cell treatment was shown to be efficacious compared to placebo. This large study of dogs also provides valuable animal clinical safety and efficacy outcome data to our colleagues developing human stem cell therapy. PMID:27695698

In situ microparticle analysis of marine phytoplankton cells with infrared laser-based optical tweezers

NASA Astrophysics Data System (ADS)

Sonek, G. J.; Liu, Y.; Iturriaga, R. H.

1995-11-01

We describe the application of infrared optical tweezers to the in situ microparticle analysis of marine phytoplankton cells. A Nd:YAG laser (lambda=3D 1064 nm) trap is used to confine and manipulate single Nannochloris and Synechococcus cells in an enriched seawater medium while spectral fluorescence and Lorenz-Mie backscatter signals are simultaneously acquired under a variety of excitation and trapping conditions. Variations in the measured fluorescence intensities of chlorophyll a (Chl a) and phycoerythrin pigments in phytoplankton cells are observed. These variations are related, in part, to basic intrasample variability, but they also indicate that increasing ultraviolet-exposure time and infrared trapping power may have short-term effects on cellular physiology that are related to Chl a photobleaching and laser-induced heating, respectively. The use of optical tweezers to study the factors that affect marine cell physiology and the processes of absorption, scattering, and attenuation by individual cells, organisms, and particulate matter that contribute to optical closure on a microscopic scale are also described. (c)1995 Optical Society of America

Mesenchymal stem cells: environmentally responsive therapeutics for regenerative medicine

PubMed Central

Murphy, Matthew B; Moncivais, Kathryn; Caplan, Arnold I

2013-01-01

Mesenchymal stem cells (MSCs) are partially defined by their ability to differentiate into tissues including bone, cartilage and adipose in vitro, but it is their trophic, paracrine and immunomodulatory functions that may have the greatest therapeutic impact in vivo. Unlike pharmaceutical treatments that deliver a single agent at a specific dose, MSCs are site regulated and secrete bioactive factors and signals at variable concentrations in response to local microenvironmental cues. Significant progress has been made in understanding the biochemical and metabolic mechanisms and feedback associated with MSC response. The anti-inflammatory and immunomodulatory capacity of MSC may be paramount in the restoration of localized or systemic conditions for normal healing and tissue regeneration. Allogeneic MSC treatments, categorized as a drug by regulatory agencies, have been widely pursued, but new studies demonstrate the efficacy of autologous MSC therapies, even for individuals affected by a disease state. Safety and regulatory concerns surrounding allogeneic cell preparations make autologous and minimally manipulated cell therapies an attractive option for many regenerative, anti-inflammatory and autoimmune applications. PMID:24232253

Dielectrophoresis-Based Sample Handling in General-Purpose Programmable Diagnostic Instruments

PubMed Central

Gascoyne, Peter R. C.; Vykoukal, Jody V.

2009-01-01

As the molecular origins of disease are better understood, the need for affordable, rapid, and automated technologies that enable microscale molecular diagnostics has become apparent. Widespread use of microsystems that perform sample preparation and molecular analysis could ensure that the benefits of new biomedical discoveries are realized by a maximum number of people, even those in environments lacking any infrastructure. While progress has been made in developing miniaturized diagnostic systems, samples are generally processed off-device using labor-intensive and time-consuming traditional sample preparation methods. We present the concept of an integrated programmable general-purpose sample analysis processor (GSAP) architecture where raw samples are routed to separation and analysis functional blocks contained within a single device. Several dielectrophoresis-based methods that could serve as the foundation for building GSAP functional blocks are reviewed including methods for cell and particle sorting, cell focusing, cell ac impedance analysis, cell lysis, and the manipulation of molecules and reagent droplets. PMID:19684877

Dnd Is a Critical Specifier of Primordial Germ Cells in the Medaka Fish.

PubMed

Hong, Ni; Li, Mingyou; Yuan, Yongming; Wang, Tiansu; Yi, Meisheng; Xu, Hongyan; Zeng, Huaqiang; Song, Jianxing; Hong, Yunhan

2016-03-08

Primordial germ cell (PGC) specification occurs early in development. PGC specifiers have been identified in Drosophila, mouse, and human but remained elusive in most animals. Here we identify the RNA-binding protein Dnd as a critical PGC specifier in the medaka fish (Oryzias latipes). Dnd depletion specifically abolished PGCs, and its overexpression boosted PGCs. We established a single-cell culture procedure enabling lineage tracing in vitro. We show that individual blastomeres from cleavage embryos at the 32- and 64-cell stages are capable of PGC production in culture. Importantly, Dnd overexpression increases PGCs via increasing PGC precursors. Strikingly, dnd RNA forms prominent particles that segregate asymmetrically. Dnd concentrates in germ plasm and stabilizes germ plasm RNA. Therefore, Dnd is a critical specifier of fish PGCs and utilizes particle partition as a previously unidentified mechanism for asymmetric segregation. These findings offer insights into PGC specification and manipulation in medaka as a lower vertebrate model. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Intact mitochondrial Ca2+ uniport is essential for agonist-induced activation of endothelial nitric oxide synthase (eNOS).

PubMed

Charoensin, Suphachai; Eroglu, Emrah; Opelt, Marissa; Bischof, Helmut; Madreiter-Sokolowski, Corina T; Kirsch, Andrijana; Depaoli, Maria R; Frank, Saša; Schrammel, Astrid; Mayer, Bernd; Waldeck-Weiermair, Markus; Graier, Wolfgang F; Malli, Roland

2017-01-01

Mitochondrial Ca 2+ uptake regulates diverse endothelial cell functions and has also been related to nitric oxide (NO • ) production. However, it is not entirely clear if the organelles support or counteract NO • biosynthesis by taking up Ca 2+ . The objective of this study was to verify whether or not mitochondrial Ca 2+ uptake influences Ca 2+ -triggered NO • generation by endothelial NO • synthase (eNOS) in an immortalized endothelial cell line (EA.hy926), respective primary human umbilical vein endothelial cells (HUVECs) and eNOS-RFP (red fluorescent protein) expressing human embryonic kidney (HEK293) cells. We used novel genetically encoded fluorescent NO • probes, the geNOps, and Ca 2+ sensors to monitor single cell NO • and Ca 2+ dynamics upon cell treatment with ATP, an inositol 1,4,5-trisphosphate (IP 3 )-generating agonist. Mitochondrial Ca 2+ uptake was specifically manipulated by siRNA-mediated knock-down of recently identified key components of the mitochondrial Ca 2+ uniporter machinery. In endothelial cells and the eNOS-RFP expressing HEK293 cells we show that reduced mitochondrial Ca 2+ uptake upon the knock-down of the mitochondrial calcium uniporter (MCU) protein and the essential MCU regulator (EMRE) yield considerable attenuation of the Ca 2+ -triggered NO • increase independently of global cytosolic Ca 2+ signals. The knock-down of mitochondrial calcium uptake 1 (MICU1), a gatekeeper of the MCU, increased both mitochondrial Ca 2+ sequestration and Ca 2+ -induced NO • signals. The positive correlation between mitochondrial Ca 2+ elevation and NO • production was independent of eNOS phosphorylation at serine 1177 . Our findings emphasize that manipulating mitochondrial Ca 2+ uptake may represent a novel strategy to control eNOS-mediated NO • production. Copyright © 2016. Published by Elsevier Inc.

Control of cancer-related signal transduction networks

NASA Astrophysics Data System (ADS)

Albert, Reka

2013-03-01

Intra-cellular signaling networks are crucial to the maintenance of cellular homeostasis and for cell behavior (growth, survival, apoptosis, movement). Mutations or alterations in the expression of elements of cellular signaling networks can lead to incorrect behavioral decisions that could result in tumor development and/or the promotion of cell migration and metastasis. Thus, mitigation of the cascading effects of such dysregulations is an important control objective. My group at Penn State is collaborating with wet-bench biologists to develop and validate predictive models of various biological systems. Over the years we found that discrete dynamic modeling is very useful in molding qualitative interaction information into a predictive model. We recently demonstrated the effectiveness of network-based targeted manipulations on mitigating the disease T cell large granular lymphocyte (T-LGL) leukemia. The root of this disease is the abnormal survival of T cells which, after successfully fighting an infection, should undergo programmed cell death. We synthesized the relevant network of within-T-cell interactions from the literature, integrated it with qualitative knowledge of the dysregulated (abnormal) states of several network components, and formulated a Boolean dynamic model. The model indicated that the system possesses a steady state corresponding to the normal cell death state and a T-LGL steady state corresponding to the abnormal survival state. For each node, we evaluated the restorative manipulation consisting of maintaining the node in the state that is the opposite of its T-LGL state, e.g. knocking it out if it is overexpressed in the T-LGL state. We found that such control of any of 15 nodes led to the disappearance of the T-LGL steady state, leaving cell death as the only potential outcome from any initial condition. In four additional cases the probability of reaching the T-LGL state decreased dramatically, thus these nodes are also possible control targets. Our collaborators validated two of these predicted control mechanisms experimentally. Our work suggests that external control of a single node can be a fruitful therapeutic strategy.

Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

PubMed

Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2015-12-01

In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.

Tissue-specific insulin signaling mediates female sexual attractiveness

PubMed Central

Arbuthnott, Devin; Rundle, Howard D.; Promislow, Daniel E. L.; Pletcher, Scott D.

2017-01-01

Individuals choose their mates so as to maximize reproductive success, and one important component of this choice is assessment of traits reflecting mate quality. Little is known about why specific traits are used for mate quality assessment nor about how they reflect it. We have previously shown that global manipulation of insulin signaling, a nutrient-sensing pathway governing investment in survival versus reproduction, affects female sexual attractiveness in the fruit fly, Drosophila melanogaster. Here we demonstrate that these effects on attractiveness derive from insulin signaling in the fat body and ovarian follicle cells, whose signals are integrated by pheromone-producing cells called oenocytes. Functional ovaries were required for global insulin signaling effects on attractiveness, and manipulations of insulin signaling specifically in late follicle cells recapitulated effects of global manipulations. Interestingly, modulation of insulin signaling in the fat body produced opposite effects on attractiveness, suggesting a competitive relationship with the ovary. Furthermore, all investigated tissue-specific insulin signaling manipulations that changed attractiveness also changed fecundity in the corresponding direction, pointing to insulin pathway activity as a reliable link between fecundity and attractiveness cues. The cues themselves, cuticular hydrocarbons, responded distinctly to fat body and follicle cell manipulations, indicating independent readouts of the pathway activity from these two tissues. Thus, here we describe a system in which female attractiveness results from an apparent connection between attractiveness cues and an organismal state of high fecundity, both of which are created by lowered insulin signaling in the fat body and increased insulin signaling in late follicle cells. PMID:28817572

Tissue-specific insulin signaling mediates female sexual attractiveness.

PubMed

Fedina, Tatyana Y; Arbuthnott, Devin; Rundle, Howard D; Promislow, Daniel E L; Pletcher, Scott D

2017-08-01

Individuals choose their mates so as to maximize reproductive success, and one important component of this choice is assessment of traits reflecting mate quality. Little is known about why specific traits are used for mate quality assessment nor about how they reflect it. We have previously shown that global manipulation of insulin signaling, a nutrient-sensing pathway governing investment in survival versus reproduction, affects female sexual attractiveness in the fruit fly, Drosophila melanogaster. Here we demonstrate that these effects on attractiveness derive from insulin signaling in the fat body and ovarian follicle cells, whose signals are integrated by pheromone-producing cells called oenocytes. Functional ovaries were required for global insulin signaling effects on attractiveness, and manipulations of insulin signaling specifically in late follicle cells recapitulated effects of global manipulations. Interestingly, modulation of insulin signaling in the fat body produced opposite effects on attractiveness, suggesting a competitive relationship with the ovary. Furthermore, all investigated tissue-specific insulin signaling manipulations that changed attractiveness also changed fecundity in the corresponding direction, pointing to insulin pathway activity as a reliable link between fecundity and attractiveness cues. The cues themselves, cuticular hydrocarbons, responded distinctly to fat body and follicle cell manipulations, indicating independent readouts of the pathway activity from these two tissues. Thus, here we describe a system in which female attractiveness results from an apparent connection between attractiveness cues and an organismal state of high fecundity, both of which are created by lowered insulin signaling in the fat body and increased insulin signaling in late follicle cells.

A two degrees-of-freedom piezoelectric single-crystal micromotor

NASA Astrophysics Data System (ADS)

Chen, Zhijiang; Li, Xiaotian; Liu, Guoxi; Dong, Shuxiang

2014-12-01

A two degrees-of-freedom (DOF) ultrasonic micromotor made of piezoelectric Pb(In1/2Nb1/2)O3-Pb(Mg1/3Nb2/3)O3-PbTiO3 (PIN-PMN-PT) single crystal square-bar (dimensions 2 × 2 × 9 mm3) was developed. The PIN-PMN-PT square-bar stator can generate standing wave elliptical motions in two orthogonal vertical planes by combining the first longitudinal and second bending vibration modes, enabling it to drive a slider in two orthogonal directions. The relatively large driving forces of 0.25 N and motion speed of 35 mm/s were obtained under a voltage of 80 Vpp at its resonance frequency of 87.5 kHz. The proposed micromotor has potential for applications in micro robots, cell manipulators, and digital cameras as a two-DOF actuator.

Non-contact fiber-optical trapping of motile bacteria: dynamics observation and energy estimation

PubMed Central

Xin, Hongbao; Liu, Qingyuan; Li, Baojun

2014-01-01

The dynamics and energy conversion of bacteria are strongly associated with bacterial activities, such as survival, spreading of bacterial diseases and their pathogenesis. Although different discoveries have been reported on trapped bacteria (i.e. immobilized bacteria), the investigation on the dynamics and energy conversion of motile bacteria in the process of trapping is highly desirable. Here, we report a non-contact optical trapping of motile bacteria using a modified tapered optical fiber. Using Escherichia coli as an example, both single and multiple motile bacteria have been trapped and manipulated in a non-contact manner. Bacterial dynamics has been observed and bacterial energy has been estimated in the trapping process. This non-contact optical trapping provides a new opportunity for better understanding the bacterial dynamics and energy conversion at the single cell level. PMID:25300713

Key Transcription Factors in the Differentiation of Mesenchymal Stem Cells

PubMed Central

Almalki, Sami G.; Agrawal, Devendra K.

2016-01-01

Mesenchymal stem cells (MSCs) are multipotent cells that represent a promising source for regenerative medicine. MSCs are capable of osteogenic, chondrogenic, adipogenic and myogenic differentiation. Efficacy of differentiated MSCs to regenerate cells in the injured tissues requires the ability to maintain the differentiation toward the desired cell fate. Since MSCs represent an attractive source for autologous transplantation, cellular and molecular signaling pathways and micro-environmental changes have been studied in order to understand the role of cytokines, chemokines, and transcription factors on the differentiation of MSCs. The differentiation of MSC into a mesenchymal lineage is genetically manipulated and promoted by specific transcription factors associated with a particular cell lineage. Recent studies have explored the integration of transcription factors, including Runx2, Sox9, PPARγ, MyoD, GATA4, and GATA6 in the differentiation of MSCs. Therefore, the overexpression of a single transcription factor in MSCs may promote trans-differentiation into specific cell lineage, which can be used for treatment of some diseases. In this review, we critically discussed and evaluated the role of transcription factors and related signaling pathways that affect the differentiation of MSCs toward adipocytes, chondrocytes, osteocytes, skeletal muscle cells, cardiomyocytes, and smooth muscle cells. PMID:27012163

Restricted exchange microenvironments for cell culture.

PubMed

Hoh, Jan H; Werbin, Jeffrey L; Heinz, William F

2018-03-01

Metabolite diffusion in tissues produces gradients and heterogeneous microenvironments that are not captured in standard 2D cell culture models. Here we describe restricted exchange environment chambers (REECs) in which diffusive gradients are formed and manipulated on length scales approximating those found in vivo. In REECs, cells are grown in 2D in an asymmetric chamber (<50 μL) formed between a coverglass and a glass bottom cell culture dish separated by a thin (~100 μm) gasket. Diffusive metabolite exchange between the chamber and bulk media occurs through one or more openings micromachined into the coverglass. Cell-generated concentration gradients form radially in REECs with a single round opening (~200 μm diameter). At steady state only cells within several hundred micrometers of the opening experience metabolite concentrations that permit survival which is analogous to diffusive exchange near a capillary in tissue. The chamber dimensions, the openings' shape, size, and number, and the cellular density and metabolic activity define the gradient structure. For example, two parallel slots above confluent cells produce the 1D equivalent of a spheroid. Using REECs, we found that fibroblasts align along the axis of diffusion while MDCK cells do not. MDCK cells do, however, exhibit significant morphological variations along the diffusive gradient.

Nicholas Metropolis Award Talk for Outstanding Doctoral Thesis Work in Computational Physics: Computational biophysics and multiscale modeling of blood cells and blood flow in health and disease

NASA Astrophysics Data System (ADS)

Fedosov, Dmitry

2011-03-01

Computational biophysics is a large and rapidly growing area of computational physics. In this talk, we will focus on a number of biophysical problems related to blood cells and blood flow in health and disease. Blood flow plays a fundamental role in a wide range of physiological processes and pathologies in the organism. To understand and, if necessary, manipulate the course of these processes it is essential to investigate blood flow under realistic conditions including deformability of blood cells, their interactions, and behavior in the complex microvascular network. Using a multiscale cell model we are able to accurately capture red blood cell mechanics, rheology, and dynamics in agreement with a number of single cell experiments. Further, this validated model yields accurate predictions of the blood rheological properties, cell migration, cell-free layer, and hemodynamic resistance in microvessels. In addition, we investigate blood related changes in malaria, which include a considerable stiffening of red blood cells and their cytoadherence to endothelium. For these biophysical problems computational modeling is able to provide new physical insights and capabilities for quantitative predictions of blood flow in health and disease.

Effects of intermittent pressure imitating rolling manipulation on calcium ion homeostasis in human skeletal muscle cells.

PubMed

Zhang, Hong; Liu, Howe; Lin, Qing; Zhang, Guohui; Mason, David C

2016-08-26

Homeostasis imbalance of intracellular Ca(2+) is one of the key pathophysiological factors in skeletal muscle injuries. Such imbalance can cause significant change in the metabolism of Ca(2+)-related biomarkers in skeletal muscle, such as superoxide dismutase (SOD), malondialdehyde (MDA) and creatine kinase (CK). Measurements of these biomarkers can be used to evaluate the degree of damage to human skeletal muscle cells (HSKMCs) injury. Rolling manipulation is the most popular myofascial release technique in Traditional Chinese Medicine. The mechanism of how this technique works in ameliorating muscle injury is unknown. This study aimed to investigate the possible Ca(2+) mediated effects of intermittent pressure imitating rolling manipulation (IPIRM) of Traditional Chinese Medicine in the injured HSKMCs. The normal HSKMCs was used as control normal group (CNG), while the injured HSKMCs were further divided into five different groups: control injured group (CIG), Rolling manipulation group (RMG), Rolling manipulation-Verapamil group (RMVG), static pressure group (SPG) and static pressure-Verapamil group (SPVG). RMG and RMVG cells were cyclically exposed to 9.5-12.5 N/cm(2) of IPIRM at a frequency of 1.0 Hz for 10 min. SPG and SPVG were loaded to a continuous pressure of 12.5 N/cm(2) for 10 min. Verapamil, a calcium antagonist, was added into the culture mediums of both RMVG and SPVG groups to block the influx of calcium ion. Compared with the CNG (normal cells), SOD activity was remarkably decreased while both MDA content and CK activity were significantly increased in the CIG (injured cells). When the injured cells were treated with the intermittent rolling manipulation pressure (RMG), the SOD activity was significantly increased and MDA content and CK activity were remarkably decreased. These effects were suppressed by adding the calcium antagonist Verapamil into the culture medium in RMVG. On the other hand, exposure to static pressure in SPG and SPVG affected neither the SOD activity nor the MDA content and CK activity in the injured muscle cells regardless of the presence of verapamil or not in the culture medium. These data suggest that the intermittent rolling pressure with the manipulation could ameliorate HSKMCs injury through a Ca(2+) dependent pathway. Static pressure did not lead to the same results.

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses.

PubMed

Whitt, Michael A; Geisbert, Thomas W; Mire, Chad E

2016-01-01

There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycle replicating VSV from plasmid DNA transfected in cell culture began a revolution in the study of recombinant VSV (rVSV). This platform can be manipulated to study the viral genetic sequences and proteins important in the virus life cycle. Additionally, foreign genes can be inserted between naturally occurring or generated start/stop signals and polyadenylation sites within the VSV genome. VSV has a tolerance for foreign gene expression which has led to numerous rVSVs reported in the literature. Of particular interest are the very effective single-dose rVSV vaccine vectors against high-containment viruses such as filoviruses, henipaviruses, and arenaviruses. Herein we describe the methods for selecting foreign antigenic genes, selecting the location within the VSV genome for insertion, generation of rVSV using reverse genetics, and proper vaccine study designs.

Cell labeling with magnetic nanoparticles: Opportunity for magnetic cell imaging and cell manipulation

PubMed Central

2013-01-01

This tutorial describes a method of controlled cell labeling with citrate-coated ultra small superparamagnetic iron oxide nanoparticles. This method may provide basically all kinds of cells with sufficient magnetization to allow cell detection by high-resolution magnetic resonance imaging (MRI) and to enable potential magnetic manipulation. In order to efficiently exploit labeled cells, quantify the magnetic load and deliver or follow-up magnetic cells, we herein describe the main requirements that should be applied during the labeling procedure. Moreover we present some recommendations for cell detection and quantification by MRI and detail magnetic guiding on some real-case studies in vitro and in vivo. PMID:24564857

BioWord: A sequence manipulation suite for Microsoft Word

PubMed Central

2012-01-01

Background The ability to manipulate, edit and process DNA and protein sequences has rapidly become a necessary skill for practicing biologists across a wide swath of disciplines. In spite of this, most everyday sequence manipulation tools are distributed across several programs and web servers, sometimes requiring installation and typically involving frequent switching between applications. To address this problem, here we have developed BioWord, a macro-enabled self-installing template for Microsoft Word documents that integrates an extensive suite of DNA and protein sequence manipulation tools. Results BioWord is distributed as a single macro-enabled template that self-installs with a single click. After installation, BioWord will open as a tab in the Office ribbon. Biologists can then easily manipulate DNA and protein sequences using a familiar interface and minimize the need to switch between applications. Beyond simple sequence manipulation, BioWord integrates functionality ranging from dyad search and consensus logos to motif discovery and pair-wise alignment. Written in Visual Basic for Applications (VBA) as an open source, object-oriented project, BioWord allows users with varying programming experience to expand and customize the program to better meet their own needs. Conclusions BioWord integrates a powerful set of tools for biological sequence manipulation within a handy, user-friendly tab in a widely used word processing software package. The use of a simple scripting language and an object-oriented scheme facilitates customization by users and provides a very accessible educational platform for introducing students to basic bioinformatics algorithms. PMID:22676326

BioWord: a sequence manipulation suite for Microsoft Word.

PubMed

Anzaldi, Laura J; Muñoz-Fernández, Daniel; Erill, Ivan

2012-06-07

The ability to manipulate, edit and process DNA and protein sequences has rapidly become a necessary skill for practicing biologists across a wide swath of disciplines. In spite of this, most everyday sequence manipulation tools are distributed across several programs and web servers, sometimes requiring installation and typically involving frequent switching between applications. To address this problem, here we have developed BioWord, a macro-enabled self-installing template for Microsoft Word documents that integrates an extensive suite of DNA and protein sequence manipulation tools. BioWord is distributed as a single macro-enabled template that self-installs with a single click. After installation, BioWord will open as a tab in the Office ribbon. Biologists can then easily manipulate DNA and protein sequences using a familiar interface and minimize the need to switch between applications. Beyond simple sequence manipulation, BioWord integrates functionality ranging from dyad search and consensus logos to motif discovery and pair-wise alignment. Written in Visual Basic for Applications (VBA) as an open source, object-oriented project, BioWord allows users with varying programming experience to expand and customize the program to better meet their own needs. BioWord integrates a powerful set of tools for biological sequence manipulation within a handy, user-friendly tab in a widely used word processing software package. The use of a simple scripting language and an object-oriented scheme facilitates customization by users and provides a very accessible educational platform for introducing students to basic bioinformatics algorithms.

Magnetic water-in-water droplet microfluidics

NASA Astrophysics Data System (ADS)

Navi, Maryam; Abbasi, Niki; Tsai, Scott

2017-11-01

Aqueous two-phase systems (ATPS) have shown to be ideal candidates for replacing the conventional water-oil systems used in droplet microfluidics. We use an ATPS of Polyethylene Glycol (PEG) and Dextran (DEX) for microfluidic generation of magnetic water-in-water droplets. As ferrofluid partitions to DEX phase, there is no significant diffusion of ferrofluid at the interface of the droplets, rendering generation of magnetic DEX droplets in a non-magnetic continuous phase of PEG possible. In this system, both phases are water-based and highly biocompatible. We microfluidically generate magnetic DEX droplets at a flow-focusing junction in a jetting regime. We sort the droplets based on their size by placing a permanent magnet downstream of the droplet generation region, and show that the deflection of droplets is in good agreement with a mathematical model. We also show that the magnetic DEX droplets can be stabilized by lysozyme and be used for separation of single cell containing water-in-water droplets. This system of magnetic water-in-water droplet manipulation may find biomedical applications such as single-cell studies and drug delivery.

MicroRNA MiR-17 retards tissue growth and represses fibronectin expression.

PubMed

Shan, Sze Wan; Lee, Daniel Y; Deng, Zhaoqun; Shatseva, Tatiana; Jeyapalan, Zina; Du, William W; Zhang, Yaou; Xuan, Jim W; Yee, Siu-Pok; Siragam, Vinayakumar; Yang, Burton B

2009-08-01

MicroRNAs (miRNAs) are single-stranded regulatory RNAs, frequently expressed as clusters. Previous studies have demonstrated that the six-miRNA cluster miR-17~92 has important roles in tissue development and cancers. However, the precise role of each miRNA in the cluster is unknown. Here we show that overexpression of miR-17 results in decreased cell adhesion, migration and proliferation. Transgenic mice overexpressing miR-17 showed overall growth retardation, smaller organs and greatly reduced haematopoietic cell lineages. We found that fibronectin and the fibronectin type-III domain containing 3A (FNDC3A) are two targets that have their expression repressed by miR-17, both in vitro and in transgenic mice. Several lines of evidence support the notion that miR-17 causes cellular defects through its repression of fibronectin expression. Our single miRNA expression assay may be evolved to allow the manipulation of individual miRNA functions in vitro and in vivo. We anticipate that this could serve as a model for studying gene regulation by miRNAs in the development of gene therapy.

An adjustable multi-scale single beam acoustic tweezers based on ultrahigh frequency ultrasonic transducer.

PubMed

Chen, Xiaoyang; Lam, Kwok Ho; Chen, Ruimin; Chen, Zeyu; Yu, Ping; Chen, Zhongping; Shung, K Kirk; Zhou, Qifa

2017-11-01

This paper reports the fabrication, characterization, and microparticle manipulation capability of an adjustable multi-scale single beam acoustic tweezers (SBAT) that is capable of flexibly changing the size of "tweezers" like ordinary metal tweezers with a single-element ultrahigh frequency (UHF) ultrasonic transducer. The measured resonant frequency of the developed transducer at 526 MHz is the highest frequency of piezoelectric single crystal based ultrasonic transducers ever reported. This focused UHF ultrasonic transducer exhibits a wide bandwidth (95.5% at -10 dB) due to high attenuation of high-frequency ultrasound wave, which allows the SBAT effectively excite with a wide range of excitation frequency from 150 to 400 MHz by using the "piezoelectric actuator" model. Through controlling the excitation frequency, the wavelength of ultrasound emitted from the SBAT can be changed to selectively manipulate a single microparticle of different sizes (3-100 μm) by using only one transducer. This concept of flexibly changing "tweezers" size is firstly introduced into the study of SBAT. At the same time, it was found that this incident ultrasound wavelength play an important role in lateral trapping and manipulation for microparticle of different sizes. Biotechnol. Bioeng. 2017;114: 2637-2647. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Interactions of light and gravity in Chara internodal cells

NASA Astrophysics Data System (ADS)

Staves, Mark P.; Whitsit, Kimberly; Yeung, Edward

2005-08-01

The "shoots" of Chara corallina are composed of large (ca. 2-5 cm length and 0.5 mm diameter) internodal cells alternating with smaller, node-forming cells. We find that these shoots are both negatively gravitropic as well as positively phototropic. Differential growth in response to both gravity and light typically takes place in the two most apical (youngest) internodal cells, however the plants can be manipulated so that all curvature takes place in a single cell. We grew Chara in aquaria filled with artificial pond water with their rhizoids in 35 mm film canisters containing soil. Thus, it was easy to reorient the axis of the plant with respect to gravity. Experimental plants were allowed to develop to a stage where they had one or two visible internodal cells. In the absence of light, internodal cells are negatively gravitropic. If gravistimulated (horizontal) internodal cells are illuminated with white light from above, gravity and light act together and more rapid curvature ensues. If however, gravistimulated internodal cells are illuminated from below, gravity and light act antagonistically and light can overcome the gravity signal. We find that gravistimulated cells illuminated from below will bend up (i.e. negatively gravitropic and negatively phototropic) at light intensities below ca. 1 μmol m-2 s-1 whereas they curve downward (positively gravitropic and positively phototropic) at higher light intensities. Preliminary studies indicate that both blue and green light stimulate phototropism whereas red light is not effective. Chara thus provides a system in which a single, statolith-free cell responds to both light and gravity and in which the interactions of the light- and gravity-induced signal transduction pathways can be investigated.

Purple sea urchin Strongylocentrotus purpuratus gamete manipulation using optical trapping and microfluidics

NASA Astrophysics Data System (ADS)

Chandsawangbhuwana, Charlie; Shi, Linda Z.; Zhu, Qingyuan; Berns, Michael W.

2013-04-01

A system has been developed that allows for optical and fluidic manipulation of gametes. The optical manipulation is performed by using a single-point gradient trap with a 40× oil immersion PH3 1.3 NA objective on a Zeiss inverted microscope. The fluidic manipulation is performed by using a custom microfluidic chamber designed to fit into the short working distance between the condenser and objective. The system is validated using purple sea urchin Strongylocentrotus purpuratus gametes and has the potential to be used for mammalian in vitro fertilization and animal husbandry.

Broadband electromagnetic dipole scattering by coupled multiple nanospheres

NASA Astrophysics Data System (ADS)

Jing, Xufeng; Ye, Qiufeng; Hong, Zhi; Zhu, Dongshuo; Shi, Guohua

2017-11-01

With the development of nanotechnology, the ability to manipulate light at the nanoscale is critical to future optical functional devices. The use of high refractive index dielectric single silicon nanoparticle can achieve electromagnetic dipole resonant properties. Compared with single nanosphere, the use of dimer and trimer introduces an additional dimension (gap size) for improving the performance of dielectric optical devices through the coupling between closely connected silicon nanospheres. When changing the gap size between the nanospheres, the interaction between the particles can be from weak to strong. Compared with single nanospheres, dimerized or trimeric nanospheres exhibit more pronounced broadband scattering properties. In addition, by introducing more complex interaction, the trimericed silicon nanospheres exhibit a more significant increase in bandwidth than expected. In addition, the presence of the substrate will also contribute to the increase in the bandwidth of the nanospheres. The broadband response in dielectric nanostructures can be effectively applied to broadband applications such as dielectric nanoantennas or solar cells.

Scalable, Lightweight, Integrated and Quick-to-Assemble (SLIQ) Hyperdrives for Functional Circuit Dissection.

PubMed

Liang, Li; Oline, Stefan N; Kirk, Justin C; Schmitt, Lukas Ian; Komorowski, Robert W; Remondes, Miguel; Halassa, Michael M

2017-01-01

Independently adjustable multielectrode arrays are routinely used to interrogate neuronal circuit function, enabling chronic in vivo monitoring of neuronal ensembles in freely behaving animals at a single-cell, single spike resolution. Despite the importance of this approach, its widespread use is limited by highly specialized design and fabrication methods. To address this, we have developed a Scalable, Lightweight, Integrated and Quick-to-assemble multielectrode array platform. This platform additionally integrates optical fibers with independently adjustable electrodes to allow simultaneous single unit recordings and circuit-specific optogenetic targeting and/or manipulation. In current designs, the fully assembled platforms are scalable from 2 to 32 microdrives, and yet range 1-3 g, light enough for small animals. Here, we describe the design process starting from intent in computer-aided design, parameter testing through finite element analysis and experimental means, and implementation of various applications across mice and rats. Combined, our methods may expand the utility of multielectrode recordings and their continued integration with other tools enabling functional dissection of intact neural circuits.

Avian Biotechnology.

PubMed

Nakamura, Yoshiaki

2017-01-01

Primordial germ cells (PGCs) generate new individuals through differentiation, maturation and fertilization. This means that the manipulation of PGCs is directly linked to the manipulation of individuals, making PGCs attractive target cells in the animal biotechnology field. A unique biological property of avian PGCs is that they circulate temporarily in the vasculature during early development, and this allows us to access and manipulate avian germ lines. Following the development of a technique for transplantation, PGCs have become central to avian biotechnology, in contrast to the use of embryo manipulation and subsequent transfer to foster mothers, as in mammalian biotechnology. Today, avian PGC transplantation combined with recent advanced manipulation techniques, including cell purification, cryopreservation, depletion, and long-term culture in vitro, have enabled the establishment of genetically modified poultry lines and ex-situ conservation of poultry genetic resources. This chapter introduces the principles, history, and procedures of producing avian germline chimeras by transplantation of PGCs, and the current status of avian germline modification as well as germplasm cryopreservation. Other fundamental avian reproductive technologies are described, including artificial insemination and embryo culture, and perspectives of industrial applications in agriculture and pharmacy are considered, including poultry productivity improvement, egg modification, disease resistance impairment and poultry gene "pharming" as well as gene banking.

High-Performance Polymer Solar Cells Based on a Wide-Bandgap Polymer Containing Pyrrolo[3,4-f]benzotriazole-5,7-dione with a Power Conversion Efficiency of 8.63.

PubMed

Lan, Liuyuan; Chen, Zhiming; Hu, Qin; Ying, Lei; Zhu, Rui; Liu, Feng; Russell, Thomas P; Huang, Fei; Cao, Yong

2016-09-01

A novel donor-acceptor type conjugated polymer based on a building block of 4,8-di(thien-2-yl) - 6-octyl-2-octyl-5 H- pyrrolo[3,4- f ]benzotriazole-5,7(6 H )-dione (TZBI) as the acceptor unit and 4,8-bis(5-(2-ethylhexyl)thiophen-2-yl)-benzo-[1,2- b :4,5- b' ]dithiophene as the donor unit, named as PTZBIBDT, is developed and used as an electron-donating material in bulk-heterojunction polymer solar cells. The resulting copolymer exhibits a wide bandgap of 1.81 eV along with relatively deep highest occupied molecular orbital energy level of -5.34 eV. Based on the optimized processing conditions, including thermal annealing, and the use of a water/alcohol cathode interlayer, the single-junction polymer solar cell based on PTZBIBDT:PC 71 BM ([6,6]-phenyl-C 71 -butyric acid methyl ester) blend film affords a power conversion efficiency of 8.63% with an open-circuit voltage of 0.87 V, a short circuit current of 13.50 mA cm -2 , and a fill factor of 73.95%, which is among the highest values reported for wide-bandgap polymers-based single-junction organic solar cells. The morphology studies on the PTZBIBDT:PC 71 BM blend film indicate that a fibrillar network can be formed and the extent of phase separation can be mani-pulated by thermal annealing. These results indicate that the TZBI unit is a very promising building block for the synthesis of wide-bandgap polymers for high-performance single-junction and tandem (or multijunction) organic solar cells.

Magnetic manipulation device for the optimization of cell processing conditions.

PubMed

Ito, Hiroshi; Kato, Ryuji; Ino, Kosuke; Honda, Hiroyuki

2010-02-01

Variability in human cell phenotypes make it's advancements in optimized cell processing necessary for personalized cell therapy. Here we propose a strategy of palm-top sized device to assist physically manipulating cells for optimizing cell preparations. For the design of such a device, we combined two conventional approaches: multi-well plate formatting and magnetic cell handling using magnetite cationic liposomes (MCLs). From our previous works, we showed the labeling applications of MCL on adhesive cells for various tissue engineering approaches. To feasibly transfer cells in multi-well plate, we here evaluated the magnetic response of MCL-labeled suspension type cells. The cell handling performance of Jurkat cells proved to be faster and more robust compared to MACS (Magnetic Cell Sorting) bead methods. To further confirm our strategy, prototype palm-top sized device "magnetic manipulation device (MMD)" was designed. In the device, the actual cell transportation efficacy of Jurkat cells was satisfying. Moreover, as a model of the most distributed clinical cell processing, primary peripheral blood mononuclear cells (PBMCs) from different volunteers were evaluated. By MMD, individual PBMCs indicated to have optimum Interleukin-2 (IL-2) concentrations for the expansion. Such huge differences of individual cells indicated that MMD, our proposing efficient and self-contained support tool, could assist the feasible and cost-effective optimization of cell processing in clinical facilities. Copyright (c) 2009 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Changes in neck pain and active range of motion after a single thoracic spine manipulation in subjects presenting with mechanical neck pain: a case series.

PubMed

Fernández-de-las-Peñas, César; Palomeque-del-Cerro, Luis; Rodríguez-Blanco, Cleofás; Gómez-Conesa, Antonia; Miangolarra-Page, Juan C

2007-05-01

Our aim was to report changes in neck pain at rest, active cervical range of motion, and neck pain at end-range of cervical motion after a single thoracic spine manipulation in a case series of patients with mechanical neck pain. Seven patients with mechanical neck pain (2 men, 5 women), 20 to 33 years old, were included. All patients received a single thoracic manipulation by an experienced manipulative therapist. The outcome measures of these cases series were neck pain at rest, as measured by a numerical pain rating scale; active cervical range of motion; and neck pain at the end of each neck motion (eg, flexion or extension). These outcomes were assessed pre treatment, 5 minutes post manipulation, and 48 hours after the intervention. A repeated-measures analysis was made with parametric tests. Within-group effect sizes were calculated using Cohen d coefficients. A significant (P < .001) decrease, with large within-group effect sizes (d > 1), in neck pain at rest were found after the thoracic spinal manipulation. A trend toward an increase in all cervical motions (flexion, extension, right or left lateral flexion, and right or left rotation) and a trend toward a decrease in neck pain at the end of each cervical motion were also found, although differences did not reach the significance (P > .05). Nevertheless, medium to large within-group effect sizes (0.5 < d < 1) were found between preintervention data and both postintervention assessments in both active range of motion and neck pain at the end of each neck motion. The present results demonstrated a clinically significant reduction in pain at rest in subjects with mechanical neck pain immediately and 48 hours following a thoracic manipulation. Although increases in all tested ranges of motion were obtained, none of them reached statistical significance at either posttreatment point. The same was found for pain at the end of range of motion for all tested ranges, with the exception of pain at the end of forward flexion at 48 hours. More than one mechanism likely explains the effects of thoracic spinal manipulation. Future controlled studies comparing spinal manipulation vs spinal mobilization of the thoracic spine are required.

DNA Free Energy Landscapes and RNA Nano-Self-Assembly Using Atomic Force Microscopy

NASA Astrophysics Data System (ADS)

Frey, Eric William

There is an important conceptual lesson which has long been appreciated by those who work in biophysics and related interdisciplinary fields. While the extraordinary behavior of biological matter is governed by its detailed atomic structure and random fluctuations, and is therefore difficult to predict, it can nevertheless be understood within simplified frameworks. Such frameworks model the system as consisting of only one or a few components, and model the behavior of the system as the occupation of a single state out of a small number of states available. The emerging widespread application of nanotechnology, such as atomic force microscopy (AFM), has expanded this understanding in eye-opening new levels of detail by enabling nano-scale control, measurement, and visualization of biological molecules. This thesis describes two independent projects, both of which illuminate this understanding using AFM, but which do so from very different perspectives. The organization of this thesis is as follows. Chapter 1 begins with an experimental background and introduction to AFM, and then describes our setup in both single-molecule manipulation and imaging modes. In Chapter 2, we describe the first project, the motivation for which is to extend methods for the experimental determination of the free energy landscape of a molecule. This chapter relies on the analysis of single-molecule manipulation data. Chapter 3 describes the second project, the motivation for which is to create RNA-based nano-structures suitable for future applications in living mammalian cells. This chapter relies mainly on imaging. Chapters 2 and 3 can thus be read and understood separately.

Molecular manipulations for enhancing luminescent bioreporters performance in the detection of toxic chemicals.

PubMed

Yagur-Kroll, Sharon; Belkin, Shimshon

2014-01-01

Microbial whole-cell bioreporters are genetically modified microorganisms that produce a quantifiable output in response to the presence of toxic chemicals or other stress factors. These bioreporters harbor a genetic fusion between a sensing element (usually a gene regulatory element responsive to the target) and a reporter element, the product of which may be quantitatively monitored either by its presence or by its activity. In this chapter we review genetic manipulations undertaken in order to improve bioluminescent bioreporter performance by increasing luminescent output, lowering the limit of detection, and shortening the response time. We describe molecular manipulations applied to all aspects of whole-cell bioreporters: the host strain, the expression system, the sensing element, and the reporter element. The molecular construction of whole-cell luminescent bioreporters, harboring fusions of gene promoter elements to reporter genes, has been around for over three decades; in most cases, these two genetic elements are combined "as is." This chapter outlines diverse molecular manipulations for enhancing the performance of such sensors.

From cells to embryos: the application of femtosecond laser pulses for altering cellular material in complex biological systems

NASA Astrophysics Data System (ADS)

Kohli, V.; Elezzabi, A. Y.

2008-02-01

We report the application of high-intensity femtosecond laser pulses as a novel tool for manipulating biological specimens. When femtosecond laser pulses were focused to a near diffraction-limited focal spot, cellular material within the laser focal volume was surgically ablated. Several dissection cuts were made in the membrane of live mammalian cells, and membrane surgery was accomplished without inducing cell collapse or disassociation. By altering how the laser pulses were applied, focal adhesions joining live epithelial cells were surgically removed, resulting in single cell isolation. To further examine the versatility of this reported tool, cells were transiently permeabilized for introducing foreign material into the cytoplasm of live mammalian cells. Localizing focused femtosecond laser pulses on the biological membrane resulted in the formation of transient pores, which were harnessed as a pathway for the delivery of exogenous material. Individual mammalian cells were permeabilized in the presence of a hyperosmotic cryoprotective disaccharide. Material delivery was confirmed by measuring the volumetric response of cells permeabilized in 0.2, 0.3, 0.4 and 0.5 M cryoprotective sugar. The survival of permeabilized cells in increasing osmolarity of sugar was assessed using a membrane integrity assay. Further demonstrating the novelty of this reported tool, laser surgery of an aquatic embryo, the zebrafish (Danio rerio), was also performed. Utilizing the transient pores that were formed in the embryonic cells of the zebrafish embryo, an exogenous fluorescent probe FITC, Streptavidin-conjugated quantum dots or plasmid DNA (sCMV) encoding EGFP was introduced into the developing embryonic cells. To determine if the laser induced any short- or long-term effects on development, laser-manipulated embryos were reared to 2 and 7 days post-fertilization and compared to control embryos at the same developmental stages. Light microscopy and scanning electron microscopy were used to compare whole body mosaics of the developed larvae. Key developmental features that were compared included the olfactory pit, dorsal, ventral and pectoral fins, notochord, otic capsule and otic vesicle. No differences in the morphology and placement of the fore-, mid- and hindbrains were observed.

Compact teleoperated laparoendoscopic single-site robotic surgical system: Kinematics, control, and operation.

PubMed

Isaac-Lowry, Oran Jacob; Okamoto, Steele; Pedram, Sahba Aghajani; Woo, Russell; Berkelman, Peter

2017-12-01

To date a variety of teleoperated surgical robotic systems have been developed to improve a surgeon's ability to perform demanding single-port procedures. However typical large systems are bulky, expensive, and afford limited angular motion, while smaller designs suffer complications arising from limited motion range, speed, and force generation. This work was to develop and validate a simple, compact, low cost single site teleoperated laparoendoscopic surgical robotic system, with demonstrated capability to carry out basic surgical procedures. This system builds upon previous work done at the University of Hawaii at Manoa and includes instrument and endoscope manipulators as well as compact articulated instruments designed to overcome single incision geometry complications. A robotic endoscope holder was used for the base, with an added support frame for teleoperated manipulators and instruments fabricated mostly from 3D printed parts. Kinematics and control methods were formulated for the novel manipulator configuration. Trajectory following results from an optical motion tracker and sample task performance results are presented. Results indicate that the system has successfully met the goal of basic surgical functionality while minimizing physical size, complexity, and cost. Copyright © 2017 John Wiley & Sons, Ltd.

Single-cell heterogeneity and cell-cycle-related viral gene bursts in the human leukaemia virus HTLV-1

PubMed Central

Billman, Martin R; Rueda, David; Bangham, Charles R M

2017-01-01

Background: The human leukaemia virus HTLV-1 expresses essential accessory genes that manipulate the expression, splicing and transport of viral mRNAs.  Two of these genes, tax and hbz, also promote proliferation of the infected cell, and both genes are thought to contribute to oncogenesis in adult T-cell leukaemia/lymphoma.  The regulation of HTLV-1 proviral latency is not understood.  tax, on the proviral plus strand, is usually silent in freshly-isolated cells, whereas the minus-strand-encoded hbz gene is persistently expressed at a low level.  However, the persistently activated host immune response to Tax indicates frequent expression of tax in vivo.  Methods: We used single-molecule RNA-FISH to quantify the expression of HTLV-1 transcripts at the single-cell level in a total of >19,000 cells from five T-cell clones, naturally infected with HTLV-1, isolated by limiting dilution from peripheral blood of HTLV-1-infected subjects.  Results: We found strong heterogeneity both within and between clones in the expression of the proviral plus-strand (detected by hybridization to the tax gene) and the minus-strand ( hbz gene). Both genes are transcribed in bursts; tax expression is enhanced in the absence of hbz, while hbz expression increased in cells with high tax expression. Surprisingly, we found that hbz expression is strongly associated with the S and G 2/M phases of the cell cycle, independent of tax expression.  Contrary to current belief, hbz is not expressed in all cells at all times, even within one clone.  In hbz-positive cells, the abundance of hbz transcripts showed a very strong positive linear correlation with nuclear volume. Conclusions: The occurrence of intense, intermittent plus-strand gene bursts in independent primary HTLV-1-infected T-cell clones from unrelated individuals strongly suggests that the HTLV-1 plus-strand is expressed in bursts in vivo.  Our results offer an explanation for the paradoxical correlations observed between the host immune response and HTLV-1 transcription. PMID:29062917

Characterization of photoactivated singlet oxygen damage in single-molecule optical trap experiments.

PubMed

Landry, Markita P; McCall, Patrick M; Qi, Zhi; Chemla, Yann R

2009-10-21

Optical traps or "tweezers" use high-power, near-infrared laser beams to manipulate and apply forces to biological systems, ranging from individual molecules to cells. Although previous studies have established that optical tweezers induce photodamage in live cells, the effects of trap irradiation have yet to be examined in vitro, at the single-molecule level. In this study, we investigate trap-induced damage in a simple system consisting of DNA molecules tethered between optically trapped polystyrene microspheres. We show that exposure to the trapping light affects the lifetime of the tethers, the efficiency with which they can be formed, and their structure. Moreover, we establish that these irreversible effects are caused by oxidative damage from singlet oxygen. This reactive state of molecular oxygen is generated locally by the optical traps in the presence of a sensitizer, which we identify as the trapped polystyrene microspheres. Trap-induced oxidative damage can be reduced greatly by working under anaerobic conditions, using additives that quench singlet oxygen, or trapping microspheres lacking the sensitizers necessary for singlet state photoexcitation. Our findings are relevant to a broad range of trap-based single-molecule experiments-the most common biological application of optical tweezers-and may guide the development of more robust experimental protocols.

Characterization of Photoactivated Singlet Oxygen Damage in Single-Molecule Optical Trap Experiments

PubMed Central

Landry, Markita P.; McCall, Patrick M.; Qi, Zhi; Chemla, Yann R.

2009-01-01

Abstract Optical traps or “tweezers” use high-power, near-infrared laser beams to manipulate and apply forces to biological systems, ranging from individual molecules to cells. Although previous studies have established that optical tweezers induce photodamage in live cells, the effects of trap irradiation have yet to be examined in vitro, at the single-molecule level. In this study, we investigate trap-induced damage in a simple system consisting of DNA molecules tethered between optically trapped polystyrene microspheres. We show that exposure to the trapping light affects the lifetime of the tethers, the efficiency with which they can be formed, and their structure. Moreover, we establish that these irreversible effects are caused by oxidative damage from singlet oxygen. This reactive state of molecular oxygen is generated locally by the optical traps in the presence of a sensitizer, which we identify as the trapped polystyrene microspheres. Trap-induced oxidative damage can be reduced greatly by working under anaerobic conditions, using additives that quench singlet oxygen, or trapping microspheres lacking the sensitizers necessary for singlet state photoexcitation. Our findings are relevant to a broad range of trap-based single-molecule experiments—the most common biological application of optical tweezers—and may guide the development of more robust experimental protocols. PMID:19843445

Horizontal Magnetic Tweezers for Micromanipulation of Single DNA-Protein Complexes

NASA Astrophysics Data System (ADS)

McAndrew, C.; Sarkar, A.; Mehl, P.

2011-03-01

We report on the development of a new magnetic force transducer or ``tweezer'' that can apply pico-Newton forces on single DNA molecules in the focus plane. Since the changes in DNA's end-to-end extension are coplanar with the pulling force, there is no need to continually refocus. The DNA constructs (λ -DNA end labeled with a 3 μ m polystyrene bead and a 2.8 μ m paramagnetic sphere) and appropriate buffer are introduced to a custom built 400 μ L to 650 μ L closed cell. This closed cell isolates our sample and produces low-noise force and extension measurements. This chamber rests on a stage fixed to a three axis micromanipulator. Entering the flat chamber are two micropipettes, a 2.5 μ m id pipette for aspirating the polystyrene bead and a 20 μ m id pipette for injecting proteins of interest. The suction and the injection pipettes are rigidly mounted to a hydraulic, three-axis micromanipulator. DNA-bead constructs, once introduced to the chamber, can be located by moving the stage over the objective. We have shown that we can easily and reputably find, capture, and manipulate single molecules of DNA within a force range of 0.1pN to 100pN.

AC electric field induced dipole-based on-chip 3D cell rotation.

PubMed

Benhal, Prateek; Chase, J Geoffrey; Gaynor, Paul; Oback, Björn; Wang, Wenhui

2014-08-07

The precise rotation of suspended cells is one of the many fundamental manipulations used in a wide range of biotechnological applications such as cell injection and enucleation in nuclear transfer (NT) cloning. Noticeably scarce among the existing rotation techniques is the three-dimensional (3D) rotation of cells on a single chip. Here we present an alternating current (ac) induced electric field-based biochip platform, which has an open-top sub-mm square chamber enclosed by four sidewall electrodes and two bottom electrodes, to achieve rotation about the two axes, thus 3D cell rotation. By applying an ac potential to the four sidewall electrodes, an in-plane (yaw) rotating electric field is generated and in-plane rotation is achieved. Similarly, by applying an ac potential to two opposite sidewall electrodes and the two bottom electrodes, an out-of-plane (pitch) rotating electric field is generated and rolling rotation is achieved. As a prompt proof-of-concept, bottom electrodes were constructed with transparent indium tin oxide (ITO) using the standard lift-off process and the sidewall electrodes were constructed using a low-cost micro-milling process and then assembled to form the chip. Through experiments, we demonstrate rotation of bovine oocytes of ~120 μm diameter about two axes, with the capability of controlling the rotation direction and the rate for each axis through control of the ac potential amplitude, frequency, and phase shift, and cell medium conductivity. The maximum observed rotation rate reached nearly 140° s⁻¹, while a consistent rotation rate reached up to 40° s⁻¹. Rotation rate spectra for zona pellucida-intact and zona pellucida-free oocytes were further compared and found to have no effective difference. This simple, transparent, cheap-to-manufacture, and open-top platform allows additional functional modules to be integrated to become a more powerful cell manipulation system.

Mesenchymal stem cells and cardiac repair

PubMed Central

Nesselmann, Catharina; Ma, Nan; Bieback, Karen; Wagner, Wolfgang; Ho, Anthony; Konttinen, Yrjö T; Zhang, Hao; Hinescu, Mihail E; Steinhoff, Gustav

2008-01-01

Accumulating clinical and experimental evidence indicates that mesenchymal stem cells (MSCs) are promising cell types in the treatment of cardiac dysfunction. They may trigger production of reparative growth factors, replace damaged cells and create an environment that favours endogenous cardiac repair. However, identifying mechanisms which regulate the role of MSCs in cardiac repair is still at work. To achieve the maximal clinical benefits, ex vivo manipulation can further enhance MSC therapeutic potential. This review focuses on the mechanism of MSCs in cardiac repair, with emphasis on ex vivo manipulation. PMID:18684237

Reevaluating the two-representation model of numerical magnitude processing.

PubMed

Jiang, Ting; Zhang, Wenfeng; Wen, Wen; Zhu, Haiting; Du, Han; Zhu, Xiangru; Gao, Xuefei; Zhang, Hongchuan; Dong, Qi; Chen, Chuansheng

2016-01-01

One debate in mathematical cognition centers on the single-representation model versus the two-representation model. Using an improved number Stroop paradigm (i.e., systematically manipulating physical size distance), in the present study we tested the predictions of the two models for number magnitude processing. The results supported the single-representation model and, more importantly, explained how a design problem (failure to manipulate physical size distance) and an analytical problem (failure to consider the interaction between congruity and task-irrelevant numerical distance) might have contributed to the evidence used to support the two-representation model. This study, therefore, can help settle the debate between the single-representation and two-representation models.

Site-selective substitutional doping with atomic precision on stepped Al (111) surface by single-atom manipulation

PubMed Central

2014-01-01

In fabrication of nano- and quantum devices, it is sometimes critical to position individual dopants at certain sites precisely to obtain the specific or enhanced functionalities. With first-principles simulations, we propose a method for substitutional doping of individual atom at a certain position on a stepped metal surface by single-atom manipulation. A selected atom at the step of Al (111) surface could be extracted vertically with an Al trimer-apex tip, and then the dopant atom will be positioned to this site. The details of the entire process including potential energy curves are given, which suggests the reliability of the proposed single-atom doping method. PMID:24899871

Site-selective substitutional doping with atomic precision on stepped Al (111) surface by single-atom manipulation.

PubMed

Chen, Chang; Zhang, Jinhu; Dong, Guofeng; Shao, Hezhu; Ning, Bo-Yuan; Zhao, Li; Ning, Xi-Jing; Zhuang, Jun

2014-01-01

In fabrication of nano- and quantum devices, it is sometimes critical to position individual dopants at certain sites precisely to obtain the specific or enhanced functionalities. With first-principles simulations, we propose a method for substitutional doping of individual atom at a certain position on a stepped metal surface by single-atom manipulation. A selected atom at the step of Al (111) surface could be extracted vertically with an Al trimer-apex tip, and then the dopant atom will be positioned to this site. The details of the entire process including potential energy curves are given, which suggests the reliability of the proposed single-atom doping method.

Modeling and control of a hydraulically actuated flexible-prismatic link robot

DOE Office of Scientific and Technical Information (OSTI.GOV)

Love, L.; Kress, R.; Jansen, J.

1996-12-01

Most of the research related to flexible link manipulators to date has focused on single link, fixed length, single plane of vibration test beds. In addition, actuation has been predominantly based upon electromagnetic motors. Ironically, these elements are rarely found in the existing industrial long reach systems. This manuscript describes a new hydraulically actuated, long reach manipulator with a flexible prismatic link at Oak Ridge National Laboratory (ORNL). Focus is directed towards both modeling and control of hydraulic actuators as well as flexible links that have variable natural frequencies.

Electromagnetically actuated micromanipulator using an acoustically oscillating bubble

NASA Astrophysics Data System (ADS)

Kwon, J. O.; Yang, J. S.; Lee, S. J.; Rhee, K.; Chung, S. K.

2011-11-01

A novel non-invasive micromanipulation technique has been developed where a microrobot swimming in an aqueous medium manipulates micro-objects, through electromagnetic actuation using an acoustically oscillating bubble attached to the microrobot as a grasping tool. This micromanipulation concept was experimentally verified; an investigation of electromagnetic actuation and acoustic excitation was also performed. Two-dimensional propulsion of a magnetic piece was demonstrated through electromagnetic actuation, using three pairs of electric coils surrounding the water chamber, and confirming that the propulsion speed of the magnetic piece was linearly proportional to the applied current intensity. Micro-object manipulation was separately demonstrated using an air bubble with glass beads (80 µm diameter) and a steel ball (800 µm diameter) in an aqueous medium. Upon acoustic excitation of the bubble by a piezo-actuator around its resonant frequency, the generated radiation force attracted and captured the neighboring glass beads and steel ball. The grasping force was indirectly measured by exposing the glass beads captured by the oscillating bubble to a stream generated by an auto-syringe pump in a mini-channel. By measuring the maximum speed of the streaming flow when the glass beads detached from the oscillating bubble and flowed downstream, the grasping force was calculated as 50 nN, based on Stokes' drag approximation. Finally, a fish egg was successfully manipulated with the integration of electromagnetic actuation and acoustic excitation, using a mini-robot consisting of a millimeter-sized magnetic piece with a bubble attached to its bottom. This novel micromanipulation may be an efficient tool for both micro device assembly and single-cell manipulation.

Contactless microparticle control via ultrahigh frequency needle type single beam acoustic tweezers

NASA Astrophysics Data System (ADS)

Fei, Chunlong; Li, Ying; Zhu, Benpeng; Chiu, Chi Tat; Chen, Zeyu; Li, Di; Yang, Yintang; Kirk Shung, K.; Zhou, Qifa

2016-10-01

This paper reports on contactless microparticle manipulation including single-particle controlled trapping, transportation, and patterning via single beam acoustic radiation forces. As the core component of single beam acoustic tweezers, a needle type ultrasonic transducer was designed and fabricated with center frequency higher than 300 MHz and -6 dB fractional bandwidth as large as 64%. The transducer was built for an f-number close to 1.0, and the desired focal depth was achieved by press-focusing technology. Its lateral resolution was measured to be better than 6.7 μm by scanning a 4 μm tungsten wire target. Tightly focused acoustic beam produced by the transducer was shown to be capable of manipulating individual microspheres as small as 3 μm. "USC" patterning with 15 μm microspheres was demonstrated without affecting nearby microspheres. These promising results may expand the applications in biomedical and biophysical research of single beam acoustic tweezers.

The amphibian egg as a model system for analyzing gravity effects

NASA Technical Reports Server (NTRS)

Malacinski, G. M.; Neff, A. W.

1989-01-01

Amphibian eggs provide several advantageous features as a model system for analyzing the effects of gravity on single cells. Those features include large size, readily tracked intracellular inclusions, and ease of experimental manipulation. Employing novel gravity orientation as a tool, a substantial data base is being developed. That information is being used to construct a three-dimensional model of the frog (Xenopus laevis) egg. Internal cytoplasmic organization (rather than surface features) are being emphasized. Several cytoplasmic compartments (domains) have been elucidated, and their behavior in inverted eggs monitored. They have been incorporated into the model, and serve as a point of departure for further inquiry and speculation.

The amphibian egg as a model system for analyzing gravity effects

NASA Astrophysics Data System (ADS)

Malacinski, G. M.; Neff, A. W.

Amphibian eggs provide several advantageous features as a model system for analyzing the effects of gravity on single cells. Those features include large size, readily tracked intracellular inclusions, and ease of experimental manipulation. Employing novel gravity orientation as a tool, a substantial data base is being developed. That information is being used to construct a 3-D model of the frog (Xenopus laevis) egg. Internal cytoplasmic organization (rather than surface features) are being emphasized. Several cytoplasmic compartments (domains) have been elucidated, and their behavior in inverted eggs monitored. They have been incorporated into the model, and serve as a point of departure for further inquiry and speculation.

Genetic Approaches to Reveal the Connectivity of Adult-Born Neurons

PubMed Central

Arenkiel, Benjamin R.

2011-01-01

Much has been learned about the environmental and molecular factors that influence the division, migration, and programmed cell death of adult-born neurons in the mammalian brain. However, detailed knowledge of the mechanisms that govern the formation and maintenance of functional circuit connectivity via adult neurogenesis remains elusive. Recent advances in genetic technologies now afford the ability to precisely target discrete brain tissues, neuronal subtypes, and even single neurons for vital reporter expression and controlled activity manipulations. Here, I review current viral tracing methods, heterologous receptor expression systems, and optogenetic technologies that hold promise toward elucidating the wiring diagrams and circuit properties of adult-born neurons. PMID:21519388

Long-term live-cell imaging reveals new roles for Salmonella effector proteins SseG and SteA.

PubMed

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

2017-01-01

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 (17 h) 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 hyper-replication 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. © 2016 John Wiley & Sons Ltd.

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

PubMed Central

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

A Review of Single Source Precursors for the Deposition of Ternary Chalcopyrite Materials

NASA Technical Reports Server (NTRS)

Banger, K. K.; Cowen, J.; Harris, J.; McClarnon, R.; Hehemann, D. G.; Duraj, S. A.; Scheiman, D.; Hepp, A. F.

2002-01-01

The development of thin-film solar cells on flexible, lightweight, space-qualified durable substrates (i.e. Kapton) provides an attractive solution to fabricating solar arrays with high specific power, (W/kg). The syntheses and thermal modulation of ternary single source precursors, based on the [{LR}2Cu(SR')2In(SR')2] architecture in good yields are described. Thermogravimetric analyses (TGA) and Low temperature Differential Scanning Caloriometry, (DSC) demonstrate that controlled manipulation of the steric and electronic properties of either the group five-donor and/or chalcogenide moiety permits directed adjustment of the thermal stability and physical properties of the precursors. TGA-Evolved Gas Analysis, confirms that single precursors decompose by the initial extrusion of the sulphide moiety, followed by the loss of the neutral donor group, (L) to release the ternary chalcopyrite matrix. X-ray diffraction studies, EDS and SEM on the non-volatile pyrolized material demonstrate that these derivatives afford single-phase CuInS2/CuInSe2 materials at low temperature. Thin-film fabrication studies demonstrate that these single source precursors can be used in a spray chemical vapor deposition process, for depositing CuInS2 onto flexible polymer substrates at temperatures less than 400 C.

Characterization of Two-Pore Channel 2 by Nuclear Membrane Electrophysiology

PubMed Central

Lee, Claire Shuk-Kwan; Tong, Benjamin Chun-Kit; Cheng, Cecily Wing-Hei; Hung, Harry Chun-Hin; Cheung, King-Ho

2016-01-01

Lysosomal calcium (Ca2+) release mediated by NAADP triggers signalling cascades that regulate many cellular processes. The identification of two-pore channel 2 (TPC2) as the NAADP receptor advances our understanding of lysosomal Ca2+ signalling, yet the lysosome is not amenable to traditional patch-clamp electrophysiology. Previous attempts to record TPC2 single-channel activity put TPC2 outside its native environment, which not reflect TPC2’s true physiological properties. To test the feasibility of using nuclear membrane electrophysiology for TPC2 channel characterization, we constructed a stable human TPC2-expressing DT40TKO cell line that lacks endogenous InsP3R and RyR (DT40TKO-hTPC2). Immunostaining revealed hTPC2 expression on the ER and nuclear envelope. Intracellular dialysis of NAADP into Fura-2-loaded DT40TKO-hTPC2 cells elicited cytosolic Ca2+ transients, suggesting that hTPC2 was functionally active. Using nuclear membrane electrophysiology, we detected a ~220 pS single-channel current activated by NAADP with K+ as the permeant ion. The detected single-channel recordings displayed a linear current-voltage relationship, were sensitive to Ned-19 inhibition, were biphasically regulated by NAADP concentration, and regulated by PKA phosphorylation. In summary, we developed a cell model for the characterization of the TPC2 channel and the nuclear membrane patch-clamp technique provided an alternative approach to rigorously investigate the electrophysiological properties of TPC2 with minimal manipulation. PMID:26838264

Rapid and continuous magnetic separation in droplet microfluidic devices

DOE PAGES

Brouzes, Eric; Kruse, Travis; Kimmerling, Robert; ...

2014-12-03

Here, we present a droplet microfluidic method to extract molecules of interest from a droplet in a rapid and continuous fashion. We accomplish this by first marginalizing functionalized super-paramagnetic beads within the droplet using a magnetic field, and then splitting the droplet into one droplet containing the majority of magnetic beads and one droplet containing the minority fraction. We quantitatively analysed the factors which affect the efficiency of marginalization and droplet splitting to optimize the enrichment of magnetic beads. We first characterized the interplay between the droplet velocity and the strength of the magnetic field and its effect on marginalization.more » We found that marginalization is optimal at the midline of the magnet and that marginalization is a good predictor of bead enrichment through splitting at low to moderate droplet velocities. Finally, we focused our efforts on manipulating the splitting profile to improve the enrichment provided by asymmetric splitting. We designed asymmetric splitting forks that employ capillary effects to preferentially extract the bead-rich regions of the droplets. Our strategy represents a framework to optimize magnetic bead enrichment methods tailored to the requirements of specific droplet-based applications. We anticipate that our separation technology is well suited for applications in single-cell genomics and proteomics. In particular, our method could be used to separate mRNA bound to poly-dT functionalized magnetic microparticles from single cell lysates to prepare single-cell cDNA libraries.« less

Controlling Viscous Fingering Using Time-Dependent Strategies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stone, Howard; Zheng, Zhong; Kim, Hyoungsoo

Control and stabilization of viscous fingering of immiscible fluids impacts a wide variety of pressure-driven multiphase flows. Here, we report theoretical and experimental results on time-dependent control strategy by manipulating the gap thickness b(t) in a lifting Hele-Shaw cell in the power-law form b(t) = b 1t 1/7. Experimental results show good quantitative agreement with the predictions of linear stability analysis. Furthermore, by choosing the value of a single time-independent control parameter we can either totally suppress the viscous fingering instability or maintain a series of non-splitting viscous fingers during the fluid displacement process. Besides the gap thickness of amore » Hele-Shaw cell, in principle, time-dependent control strategies can also be placed on the injection rate, viscosity of the displaced fluid, and interfacial tensions between the two fluids.« less

Imaging modes of atomic force microscopy for application in molecular and cell biology.

PubMed

Dufrêne, Yves F; Ando, Toshio; Garcia, Ricardo; Alsteens, David; Martinez-Martin, David; Engel, Andreas; Gerber, Christoph; Müller, Daniel J

2017-04-06

Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.

Controlling Viscous Fingering Using Time-Dependent Strategies

DOE PAGES

Stone, Howard; Zheng, Zhong; Kim, Hyoungsoo

2015-10-20

Control and stabilization of viscous fingering of immiscible fluids impacts a wide variety of pressure-driven multiphase flows. Here, we report theoretical and experimental results on time-dependent control strategy by manipulating the gap thickness b(t) in a lifting Hele-Shaw cell in the power-law form b(t) = b 1t 1/7. Experimental results show good quantitative agreement with the predictions of linear stability analysis. Furthermore, by choosing the value of a single time-independent control parameter we can either totally suppress the viscous fingering instability or maintain a series of non-splitting viscous fingers during the fluid displacement process. Besides the gap thickness of amore » Hele-Shaw cell, in principle, time-dependent control strategies can also be placed on the injection rate, viscosity of the displaced fluid, and interfacial tensions between the two fluids.« less

Geminiviruses for biotechnology: the art of parasite taming.

PubMed

Lozano-Durán, Rosa

2016-04-01

Viruses are intracellular pathogens that have evolved efficient strategies for replication and expression of their proteins in the host cells. Geminiviruses - plant viruses with small circular single-stranded DNA genomes - effectively manipulate plant cell processes for viral functions, entailing great potential for biotechnological applications. This potentiality has been realized in the form of protein expression and gene-silencing vectors, and, more recently, vectors for genome editing - a technology that these viruses seem particularly well-suited to facilitate. This insight offers an overview of the biological properties of geminiviruses, with emphasis on those leveraging development of geminivirus-based replicons. It illustrates the basis for engineering geminivirus-based replicons and their applications. Furthermore, it discusses the reported use and future perspectives of geminivirus-based replicons for genome editing. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

18. Photocopy of photograph. VIEW WITHIN POSTMORTEM CELL OF MANIPULATOR ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

18. Photocopy of photograph. VIEW WITHIN POST-MORTEM CELL OF MANIPULATOR ARMS BEING USED TO MOVE METAL BARS FROM ONE LOCATION TO ANOTHER. Photographer unknown, ca. 1965, original photograph and negative on file at the Remote Sensing Laboratory, Department of Energy, Nevada Operations Office. - Nevada Test Site, Engine Maintenance Assembly & Disassembly Facility, Area 25, Jackass Flats, Mercury, Nye County, NV

Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules.

PubMed

Greulich, K O; Pilarczyk, G; Hoffmann, A; Meyer Zu Hörste, G; Schäfer, B; Uhl, V; Monajembashi, S

2000-06-01

Complete manipulation by laser light allows precise and gentle treatment of plant cells, subcellular structures, and even individual DNA molecules. Recently, affordable lasers have become available for the construction of microbeams as well as for optical tweezers. This may generate new interest in these tools for plant biologists. Early experiments, reviewed in this journal, showed that laser supported microinjection of material into plant cells or tissues circumvents mechanical problems encountered in microinjection by fragile glass capillaries. Plant protoplasts could be fused with each other when under microscopical observation, and it was no major problem to generate a triple or quadruple fusion product. In the present paper we review experiments where membrane material was prepared from root hair tips and microgravity was simulated in algae. As many plant cells are transparent, it is possible to work inside living, intact cells. New experiments show that it is possible to release by optical micromanipulation, with high spatial resolutions, intracellular calcium from caged compounds and to study calcium oscillations. An example for avian cardiac tissue is given, but the technique is also suitable for plant cell research. As a more technical tool, optical tweezers can be used to spatially fix subcellular structures otherwise moving inside a cell and thus make them available for investigation with a confocal microscope even when the time for image formation is extended (for example at low fluorescence emission). A molecular biological example is the handling of chromosomes and isolated individual DNA molecules by laser microtools. For example, chromosomes can be cut along complex trajectories, not only perpendicular to their long axis. Single DNA molecules are cut by the laser microbeam and, after coupling such a molecule to a polystrene microbead, are handled in complex geometries. Here, the individual DNA molecules are made visible with a conventional fluorescence microscope by fluorescent dyes such as SYBRGreen. The cutting of a single DNA molecule by molecules of the restriction endonuclease EcoRI can be observed directly, i.e. a type of single molecule restriction analysis is possible. Finally, mechanical properties of individual DNA molecules can be observed directly.

Hybrid microfluidics combined with active and passive approaches for continuous cell separation.

PubMed

Yan, Sheng; Zhang, Jun; Yuan, Dan; Li, Weihua

2017-01-01

Microfluidics, which is classified as either active or passive, is capable of separating cells of interest from a complex and heterogeneous sample. Active methods utilise external fields such as electric, magnetic, acoustic, and optical to drive cells for separation, while passive methods utilise channel structures, intrinsic hydrodynamic forces, and steric hindrances to manipulate cells. However, when processing complex biological samples such as whole blood with rare cells, separation with a single module microfluidic device is difficult. Hybrid microfluidics is an emerging technique, which utilises active and passive methods whilst fulfilling higher requirements for stable performance, versatility, and convenience, including (i) the ability to process multi-target cells, (ii) enhanced ability for multiplexed separation, (iii) higher sensitivity, and (iv) tunability for a wider operational range. This review introduces the fundamental physics and typical formats for subclasses of hybrid microfluidic devices based on their different physical fields; presents current examples of cell sorting to highlight the advantage and usefulness of hybrid microfluidics on biomedicine, and then discusses the challenges and perspective of future development and the promising direction of research in this field. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Investigating cell mechanics with atomic force microscopy

PubMed Central

Haase, Kristina; Pelling, Andrew E.

2015-01-01

Transmission of mechanical force is crucial for normal cell development and functioning. However, the process of mechanotransduction cannot be studied in isolation from cell mechanics. Thus, in order to understand how cells ‘feel’, we must first understand how they deform and recover from physical perturbations. Owing to its versatility, atomic force microscopy (AFM) has become a popular tool to study intrinsic cellular mechanical properties. Used to directly manipulate and examine whole and subcellular reactions, AFM allows for top-down and reconstitutive approaches to mechanical characterization. These studies show that the responses of cells and their components are complex, and largely depend on the magnitude and time scale of loading. In this review, we generally describe the mechanotransductive process through discussion of well-known mechanosensors. We then focus on discussion of recent examples where AFM is used to specifically probe the elastic and inelastic responses of single cells undergoing deformation. We present a brief overview of classical and current models often used to characterize observed cellular phenomena in response to force. Both simple mechanistic models and complex nonlinear models have been used to describe the observed cellular behaviours, however a unifying description of cell mechanics has not yet been resolved. PMID:25589563

Concise review: programming human pluripotent stem cells into blood.

PubMed

Easterbrook, Jennifer; Fidanza, Antonella; Forrester, Lesley M

2016-06-01

Blood disorders are treated with cell therapies including haematopoietic stem cell (HSC) transplantation as well as platelet and red blood cell transfusions. However the source of cells is entirely dependent on donors, procedures are susceptible to transfusion-transmitted infections and serious complications can arise in recipients due to immunological incompatibility. These problems could be alleviated if it was possible to produce haematopoietic cells in vitro from an autologous and renewable cell source. The production of haematopoietic cells in the laboratory from human induced pluripotent stem cells (iPSCs) may provide a route to realize this goal but it has proven challenging to generate long-term reconstituting HSCs. To date, the optimization of differentiation protocols has mostly relied on the manipulation of extrinsic signals to mimic the in vivo environment. We review studies that have taken an alternative approach to modulate intrinsic signals by enforced expression of transcription factors. Single and combinations of multiple transcription factors have been used in a variety of contexts to enhance the production of haematopoietic cells from human pluripotent stem cells. This programming approach, together with the recent advances in the production and use of synthetic transcription factors, holds great promise for the production of fully functional HSCs in the future. © 2016 The Authors. British Journal of Haematology published by John Wiley & Sons Ltd.

A simple method for multiday imaging of slice cultures.

PubMed

Seidl, Armin H; Rubel, Edwin W

2010-01-01

The organotypic slice culture (Stoppini et al. A simple method for organotypic cultures of nervous tissue. 1991;37:173-182) has become the method of choice to answer a variety of questions in neuroscience. For many experiments, however, it would be beneficial to image or manipulate a slice culture repeatedly, for example, over the course of many days. We prepared organotypic slice cultures of the auditory brainstem of P3 and P4 mice and kept them in vitro for up to 4 weeks. Single cells in the auditory brainstem were transfected with plasmids expressing fluorescent proteins by way of electroporation (Haas et al. Single-cell electroporation for gene transfer in vivo. 2001;29:583-591). The culture was then placed in a chamber perfused with oxygenated ACSF and the labeled cell imaged with an inverted wide-field microscope repeatedly for multiple days, recording several time-points per day, before returning the slice to the incubator. We describe a simple method to image a slice culture preparation during the course of multiple days and over many continuous hours, without noticeable damage to the tissue or photobleaching. Our method uses a simple, inexpensive custom-built insulator constructed around the microscope to maintain controlled temperature and uses a perfusion chamber as used for in vitro slice recordings. (c) 2009 Wiley-Liss, Inc.

Engineering cortical neuron polarity with nanomagnets on a chip.

PubMed

Kunze, Anja; Tseng, Peter; Godzich, Chanya; Murray, Coleman; Caputo, Anna; Schweizer, Felix E; Di Carlo, Dino

2015-01-01

Intra- and extracellular signaling play critical roles in cell polarity, ultimately leading to the development of functional cell-cell connections, tissues, and organs. In the brain, pathologically oriented neurons are often the cause for disordered circuits, severely impacting motor function, perception, and memory. Aside from control through gene expression and signaling pathways, it is known that nervous system development can be manipulated by mechanical stimuli (e.g., outgrowth of axons through externally applied forces). The inverse is true as well: intracellular molecular signals can be converted into forces to yield axonal outgrowth. The complete role played by mechanical signals in mediating single-cell polarity, however, remains currently unclear. Here we employ highly parallelized nanomagnets on a chip to exert local mechanical stimuli on cortical neurons, independently of the amount of superparamagnetic nanoparticles taken up by the cells. The chip-based approach was utilized to quantify the effect of nanoparticle-mediated forces on the intracellular cytoskeleton as visualized by the distribution of the microtubule-associated protein tau. While single cortical neurons prefer to assemble tau proteins following poly-L-lysine surface cues, an optimal force range of 4.5-70 pN by the nanomagnets initiated a tau distribution opposed to the pattern cue. In larger cell clusters (groups comprising six or more cells), nanoparticle-mediated forces induced tau repositioning in an observed range of 190-270 pN, and initiation of magnetic field-directed cell displacement was observed at forces above 300 pN. Our findings lay the groundwork for high-resolution mechanical encoding of neural networks in vitro, mechanically driven cell polarization in brain tissues, and neurotherapeutic approaches using functionalized superparamagnetic nanoparticles to potentially restore disordered neural circuits.

Six cloned calves produced from adult fibroblast cells after long-term culture

PubMed Central

Kubota, Chikara; Yamakuchi, Hiroshi; Todoroki, Junichi; Mizoshita, Kazunori; Tabara, Norio; Barber, Michele; Yang, Xiangzhong

2000-01-01

Cloning whole animals with somatic cells as parents offers the possibility of targeted genetic manipulations in vitro such as “gene knock-out” by homologous recombination. However, such manipulation requires prolonged culture of nuclear donor cells. Previous successes in cloning have been limited to the use of cells collected either fresh or after short-term culture. Therefore, demonstration of genetic totipotency of cells after prolonged culture is pivotal to combining site-specific genetic manipulations and cloning. Here we report birth of six clones of an aged (17-year-old) Japanese Black Beef bull using ear skin fibroblast cells as nuclear donor cells after up to 3 months of in vitro culture (10–15 passages). We observed higher developmental rates for embryos derived from later passages (10 and 15) as compared with those embryos from an early passage (passage 5). The four surviving clones are now 10–12 months of age and appear normal, similar to their naturally reproduced peers. These data show that fibroblasts of aged animals remain competent for cloning, and prolonged culture does not affect the cloning competence of adult somatic donor cells. PMID:10655472

Voltage signals of individual Purkinje cell dendrites in rat cerebellar slices.

PubMed

Borst, A; Heck, D; Thomann, M

1997-11-28

For investigating neuronal information processing at the cellular level, a technique which visualizes the voltage distribution within single neurons in situ would be extremely useful. Voltage-sensitive dyes are, in principle, capable of reporting membrane potential [Cohen, L.B. and Salzberg, B.M., Rev. Physiol. Biochem. Pharmacol., 83 (1978) 35-88; Grinvald, A., Lieke, E.E., Frostig, R.D. and Hildesheim, R., J. Neurosci., 14 (1994) 2545-2568; Kleinfeld, D., Delaney, K.R., Fee, M.S., Flores, J.A., Tank, D.W. and Gelperin, A., J. Neurophysiol., 72 (1994) 1402-1419]. However, their application to single cells internally is technically difficult [Antic, S. and Zecevic, D., J. Neurosci., 15 (1995) 1392-1405; Grinvald, A., Salzberg, B.M., Lev-Ram, V. and Hildesheim, R., Biophys. J., 51 (1987) 643-651; Kogan, A., Ross, W.N., Zecevic, D. and Lasser-Ross, N., Brain Res., 700 (1995) 235-239; Zecevic, D., Nature, 381 (1996) 322-325]. An alternative strategy consists in applying the dye from the outside to all cells in the tissue, while manipulating a single cell by current injection [Krauthamer, V. and Ross, W.N., J. Neurosci., 4 (1984) 673-682; Ross, W.N. and Krauthamer, V., J. Neurosci., 4 (1984) 659-672]. Here, we modify this technique to further enhance spatial at the cost of temporal resolution [Borst, A., Z. Naturforsch., 50 (1995) 435-438]. Applied to rat cerebellar slices we demonstrate that the potential spread in individual Purkinje cells can be imaged up to even fine dendritic branches. The acquired optical signals suggest that steadily hyperpolarized Purkinje cells are electrically compact. When permanently depolarized, the somatic input resistance is significantly diminished, yet the spatial voltage drop along the dendrites remains unchanged. As demonstrated by compartmental modeling, this hints to a concentration of outward rectifying currents at the soma of the cells.

Mobile Manipulation and Mobility as Manipulation: Design and Algorithms of RoboSimian

DTIC Science & Technology

2014-05-01

feature was left out for the competition hands. The hand has three under-actuated fingers, each with a braided Dyneema R© tendon wrapped around pulleys at...models of objects (e.g. valves, ladders, hoses , etc.) into the world manually so that RoboSimian could interact with objects for manipulation. The remote...with a single button press (e.g. “rotate-valve”, “insert- hose ”, “push-open-door”), depending on the task. Note that since the plan module was run on

Single-step generation of gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9.

PubMed

Matsunaga, Taichi; Yamashita, Jun K

2014-02-07

Specific gene knockout and rescue experiments are powerful tools in developmental and stem cell biology. Nevertheless, the experiments require multiple steps of molecular manipulation for gene knockout and subsequent rescue procedures. Here we report an efficient and single step strategy to generate gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9 genome editing technology. We inserted a tetracycline-regulated inducible gene promoter (tet-OFF/TRE-CMV) upstream of the endogenous promoter region of vascular endothelial growth factor receptor 2 (VEGFR2/Flk1) gene, an essential gene for endothelial cell (EC) differentiation, in mouse embryonic stem cells (ESCs) with homologous recombination. Both homo- and hetero-inserted clones were efficiently obtained through a simple selection with a drug-resistant gene. The insertion of TRE-CMV promoter disrupted endogenous Flk1 expression, resulting in null mutation in homo-inserted clones. When the inserted TRE-CMV promoter was activated with doxycycline (Dox) depletion, Flk1 expression was sufficiently recovered from the downstream genomic Flk1 gene. Whereas EC differentiation was almost completely perturbed in homo-inserted clones, Flk1 rescue with TRE-CMV promoter activation restored EC appearance, indicating that phenotypic changes in EC differentiation can be successfully reproduced with this knockout-rescue system. Thus, this promoter insertion strategy with CRISPR/Cas9 would be a novel attractive method for knockout-rescue experiments. Copyright © 2014 Elsevier Inc. All rights reserved.

Self-Rotation of Cells in an Irrotational AC E-Field in an Opto-Electrokinetics Chip

PubMed Central

Chau, Long-Ho; Liang, Wenfeng; Cheung, Florence Wing Ki; Liu, Wing Keung; Li, Wen Jung; Chen, Shih-Chi; Lee, Gwo-Bin

2013-01-01

The use of optical dielectrophoresis (ODEP) to manipulate microparticles and biological cells has become increasingly popular due to its tremendous flexibility in providing reconfigurable electrode patterns and flow channels. ODEP enables the parallel and free manipulation of small particles on a photoconductive surface on which light is projected, thus eliminating the need for complex electrode design and fabrication processes. In this paper, we demonstrate that mouse cells comprising melan-a cells, RAW 267.4 macrophage cells, peripheral white blood cells and lymphocytes, can be manipulated in an opto-electrokinetics (OEK) device with appropriate DEP parameters. Our OEK device generates a non-rotating electric field and exerts a localized DEP force on optical electrodes. Hitherto, we are the first group to report that among all the cells investigated, melan-a cells, lymphocytes and white blood cells were found to undergo self-rotation in the device in the presence of a DEP force. The rotational speed of the cells depended on the voltage and frequency applied and the cells' distance from the optical center. We discuss a possible mechanism for explaining this new observation of induced self-rotation based on the physical properties of cells. We believe that this rotation phenomenon can be used to identify cell type and to elucidate the dielectric and physical properties of cells. PMID:23320067

Establishment of novel detection system for embryonic stem cell-derived hepatocyte-like cells based on nongenetic manipulation with indocyanine green.

PubMed

Yoshie, Susumu; Ito, Jun; Shirasawa, Sakiko; Yokoyama, Tadayuki; Fujimura, Yuu; Takeda, Kazuo; Mizuguchi, Masahiro; Matsumoto, Ken; Tomotsune, Daihachiro; Sasaki, Katsunori

2012-01-01

Hepatocytes derived from embryonic stem cells (ESCs) are expected to be useful for basic research and clinical applications. However, in several studies, genetic methods used to detect and obtain them are difficult and pose major safety problems. Therefore, in this study, we established a novel detection system for hepatocytes by using indocyanine green (ICG), which is selectively taken up by hepatocytes, based on nongenetic manipulation. ICG has maximum light absorption near 780 nm, and it fluoresces between 800 and 900 nm. Making use of these properties, we developed flow cytometry equipped with an excitation lazer of 785 nm and specific bandpass filters and successfully detected ESC-derived ICG-positive cells that were periodic acid-Schiff positive and expressed hepatocyte phenotypic mRNAs. These results demonstrate that this detection system based on nongenetic manipulation with ICG will lead to isolate hepatocytes generated from ESCs and provide the appropriate levels of stability, quality, and safety required for cell source for cell-based therapy and pharmaceutical studies such as toxicology.

Nanobiotechnology for the capture and manipulation of circulating tumor cells.

PubMed

Hughes, Andrew D; King, Michael R

2012-01-01

A necessary step in metastasis is the dissemination of malignant cells into the bloodstream, where cancer cells travel throughout the body as circulating tumor cells (CTC) in search of an opportunity to seed a secondary tumor. CTC represent a valuable diagnostic tool: evidence indicates that the quantity of CTC in the blood has been shown to relate to the severity of the illness, and samples are readily obtained through routine blood draws. As such, there has been a push toward developing technologies to reliably detect CTC using a variety of molecular and immunocytochemical techniques. In addition to their use in diagnostics, CTC detection systems that isolate CTC in such a way that the cells remain viable will allow for the performance of live-cell assays to facilitate the development of personalized cancer therapies. Moreover, techniques for the direct manipulation of CTC in circulation have been developed, intending to block metastasis in situ. We review a number of current and emerging micro- and nanobiotechnology approaches for the detection, capture, and manipulation of rare CTC aimed at advancing cancer treatment. Copyright © 2011 Wiley Periodicals, Inc.

Interactions between Exosomes from Breast Cancer Cells and Primary Mammary Epithelial Cells Leads to Generation of Reactive Oxygen Species Which Induce DNA Damage Response, Stabilization of p53 and Autophagy in Epithelial Cells

PubMed Central

Dutta, Sujoy; Warshall, Case; Bandyopadhyay, Chirosree; Dutta, Dipanjan; Chandran, Bala

2014-01-01

Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive “niches”. Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7), representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs). Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS) and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC) led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR) responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment. PMID:24831807