共查询到20条相似文献,搜索用时 31 毫秒
1.
We present the first results obtained with a new instrument designed and built to study DNA-protein interactions at the single molecule level. This microscope combines optical tweezers with scanning probe microscopy and allows us to locate DNA-binding proteins on a single suspended DNA molecule. A single DNA molecule is stretched taut using the optical tweezers, while a probe is scanned along the molecule. Interaction forces between the probe and the sample are measured with the optical tweezers. The instrument thus enables us to correlate mechanical and functional properties of bound proteins with the tension within the DNA molecule. The typical friction force between a micropipette used as probe and a naked DNA molecule was found to be <1 pN. A 16 micro m DNA molecule with approximately 10-15 digoxygenin (DIG) molecules located over a 90 nm range in the middle of the DNA was used as a model system. By scanning with an antidigoxygenin (alpha-DIG) antibody-coated pipette we were able to localize these sites by exploiting the high binding affinity between this antibody-antigen pair. The estimated experimental resolution assuming an infinitesimally thin and rigid probe and a single alpha-DIG/DIG bond was 15 nm. 相似文献
2.
Otto O Steinbock LJ Wong DW Gornall JL Keyser UF 《The Review of scientific instruments》2011,82(8):086102
We have developed optical tweezers, with force measurements based on fast video tracking, for analysis and control of DNA translocation through nanocapillaries. Nanocapillaries are single-molecule biosensors with very similar characteristics to solid-state nanopores. Our novel experimental setup allows for ionic-current measurements in which the nanocapillary is oriented perpendicular to the trapping laser. Using video-based particle tracking, we are able to measure the position of DNA coated colloids at sub-millisecond resolution and in real-time. We present the first electrophoretic force and simultaneous ionic-current measurements of a single DNA molecule inside the orifice of a nanocapillary. 相似文献
3.
Sischka A Kleimann C Hachmann W Schäfer MM Seuffert I Tönsing K Anselmetti D 《The Review of scientific instruments》2008,79(6):063702
We introduce a versatile and high precision three-dimensional optical tweezers setup with minimal optical interference to measure small forces and manipulate single molecules in the vicinity of a weak reflective surface. Our tweezers system integrates an inverted optical microscope with a single IR-laser beam that is spatially filtered in an appropriate way to allow force measurements in three dimensions with remarkably high precision when operated in backscattered light detection mode. The setup was tested by overstretching a lambda-DNA in x and z directions (perpendicular and along the optical axis), and by manipulating individual lambda-DNA molecules in the vicinity of a nanopore that allowed quantitative single molecule threading experiments with minimal optical interference. 相似文献
4.
光镊技术已经成功应用于光学和微功能器件,但是,对光镊驱动复杂微转子所建模型尚不成熟。为了分析光镊的光驱动力和力矩,基于矩量法建立了一种新模型。基于此种模型的分析结果表明改变环境参量是提高光镊工作效率的方法之一,如微转子转动速率与光镊的激光功率成正比,与束腰半径成非线性关系。另一种可以大幅度提高光镊工作效率的方法是改变微转子的形状,数据表明"万字"形微转子的转动效率是相同尺寸的"十字"形转子转动效率的10-7倍。此外,通过解析力场的分布状态,可得到光压力的主要作用面,为今后的微转子设计提供依据。新模型的另一个优点是耗费时间较少,对于模拟光镊驱动微功能器件具有通用性和柔性。 相似文献
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Chan Hyuk Nam Dongjin Lee Daehie Hong Jaewon Chung 《International Journal of Precision Engineering and Manufacturing》2009,10(5):45-51
Various nano devices such as nanotubes, nanorods, nanoribbon, and nanowires have been extensively studied, since they are
the essential elements to build nanoelectronic circuits, nanochemical sensors, optical switches, etc. However, because the
nano devices are very small in size and have different shapes, it is virtually impossible to manipulate them with conventional
methods. This paper discusses the feasibility of using optical tweezers as a tool for manipulating such nano devices. The
optical tweezers developed in this paper consist of two telescopes, a FSM (Fast Steering Mirror), and a control unit, such
that multiple nano devices could be stably trapped at the specimen plane by rapidly rotating the FSM using the time sharing
method. Through the experiments of manipulating the multiple micro spheres and nanorods in various ways, it was proven that
the developed optical tweezers can be used as a versatile tool for manipulating nano devices and fabricating nano sensors. 相似文献
7.
The quantitative measurement of particle motion in optical tweezers is an important tool in the study of microrheology and can be used in a variety of scientific and industrial applications. Active microheology, in which the response of optically trapped particles to external driving forces is measured, is particularly useful in probing nonlinear viscoelastic behavior in complex fluids. Currently such experiments typically require independent measurements of the driving force and the trapped particle's response to be carefully synchronized, and therefore the experiments normally require analog equipment. In this paper we describe both a specialized camera and an imaging technique which make high-speed video microscopy a suitable tool for performing such measurements, without the need for separate measurement systems and synchronization. The use of a high-speed tracking camera based on a field programmable gate array to simultaneously track multiple particles is reported. By using this camera to simultaneously track one microsphere fixed to the wall of a driven sample chamber and another held in an optical trap, we demonstrate simultaneous optical measurement of the driving motion and the trapped probe particle response using a single instrument. Our technique is verified experimentally by active viscosity measurements on water-ethylene glycol mixtures using a phase-shift technique. 相似文献
8.
Choy JL Parekh SH Chaudhuri O Liu AP Bustamante C Footer MJ Theriot JA Fletcher DA 《The Review of scientific instruments》2007,78(4):043711
Force microscopy techniques including optical trapping, magnetic tweezers, and atomic force microscopy (AFM) have facilitated quantification of forces and distances on the molecular scale. However, sensitivity and stability limitations have prevented the application of these techniques to biophysical systems that generate large forces over long times, such as actin filament networks. Growth of actin networks drives cellular shape change and generates nano-Newtons of force over time scales of minutes to hours, and consequently network growth properties have been difficult to study. Here, we present an AFM-based differential force microscope with integrated epifluorescence imaging in which two adjacent cantilevers on the same rigid support are used to provide increased measurement stability. We demonstrate 14 nm displacement control over measurement times of 3 hours and apply the instrument to quantify actin network growth in vitro under controlled loads. By measuring both network length and total network fluorescence simultaneously, we show that the average cross-sectional density of the growing network remains constant under static loads. The differential force microscope presented here provides a sensitive method for quantifying force and displacement with long time-scale stability that is useful for measurements of slow biophysical processes in whole cells or in reconstituted molecular systems in vitro. 相似文献
9.
Micromanipulation by laser microbeam and optical tweezers: from plant cells to single molecules 总被引:2,自引:0,他引:2
K. O. Greulich G. Pilarczyk A. Hoffmann G. Meyer Zu Hörste B. Schäfer V. Uhl S. Monajembashi 《Journal of microscopy》2000,198(3):182-187
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. 相似文献
10.
Qian F Ermilov S Murdock D Brownell WE Anvari B 《The Review of scientific instruments》2004,75(9):2937-2942
We have designed and implemented a novel experimental setup which combines optical tweezers with patch-clamp apparatus to investigate the electromechanical properties of cellular plasma membranes. In this system, optical tweezers provide measurement of forces at piconewton scale, and the patch-clamp technique allows control of the cell transmembrane potential. A micron-size bead trapped by the optical tweezers is brought in contact with the membrane of a voltage-clamped cell, and subsequently moved away to form a plasma membrane tether. Bead displacement from the trapping center is monitored by a quadrant photodetector for dynamic measurements of tether force. Fluorescent beads and the corresponding fluorescence imaging optics are used to eliminate the shadow of the cell projected on the quadrant photodetector. Salient information associated with the mechanical properties of the membrane tether can thus be obtained. A unique feature of this setup is that the patch-clamp headstage and the manipulator for the recording pipette are mounted on a piezoelectric stage, preventing relative movements between the cell and the patch pipette during the process of tether pulling. Tethers can be pulled from the cell membrane at different holding potentials, and the tether force response can be measured while changing transmembrane potential. Experimental results from mammalian cochlear outer hair cells and human embryonic kidney cells are presented. 相似文献
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Construction and testing of a nanomachining instrument 总被引:9,自引:0,他引:9
Wei Gao Robert J. Hocken John A. Patten John Lovingood Don A. Lucca 《Precision Engineering》2000,24(4):320-328
This paper presents a nanomachining instrument that was developed for conducting nanocutting, nanoscratching, and nanoindentation experiments. A piezoelectric tube scanner (PZT) is employed to generate three-dimensional machining motions. The sample is moved by the PZT, and the tool is kept stationary during machining. The machining forces are measured by force sensors with a resolution of sub-milliNewtons. The instrument is compact and can be used inside optical microscopes and scanning electron microscopes. In this paper, depth-sensing indentation experiments were performed to test the basic performance of the instrument. The indentation displacement was measured by a capacitance probe situated inside the PZT tube. An experimental system was constructed to locate and image indentations. The system consists of a high magnification microscope to measure coordinates of the indentation relative to a reference corner point on the sample, and an AFM equipped with an on-axis optical imaging system for locating the indentation. A technique was also employed to establish the tool-sample contact to nanometer accuracy. Indentation experiments were carried out on three kinds of materials with different hardness. Experimental results demonstrated the instrument has the ability of performing depth-sensing indentations. The frame compliance was also evaluated from the indentation results. 相似文献
14.
Optical tweezers use focused laser light to manipulate microscopic particles. We discuss the underlying physics of the technique in terms of a gradient force exerted by the light on the particles. The versatility of optical tweezers is highlighted, in particular, we explain how spatial light modulators and various imaging methods have greatly enhanced their range of applications. 相似文献
15.
S. Bayoudh M. Mehta† H. Rubinsztein-Dunlop N. R. Heckenberg & C. Critchley† 《Journal of microscopy》2001,203(2):214-222
This paper describes experiments using optical tweezers to probe chloroplast arrangement, shape and consistency in cells of living leaf tissue and in suspension. Dual optical tweezers provided two-point contact on a single chloroplast or two-point contact on two adhered chloroplasts for manipulation in suspension. Alternatively, a microstirrer consisting of a birefringent particle trapped in an elliptically polarized laser trap was used to induce motion and tumbling of a selected chloroplast suspended in a solution. We demonstrate that displacement of chloroplasts inside the cell is extremely difficult, presumably due to chloroplast adhesion to the cytoskeleton and connections between organelles. The study also confirms that the chloroplasts are very thin and extremely cup-shaped with a concave inner surface and a convex outer surface. 相似文献
16.
A dynamic optical tweezers system is employed for generation of an optical trap in continuous rotation for manipulating a biological cell in an aqueous solution. When the rotating speed is increased, the trapped cell experiences an augmented viscous drag force, and eventually it escapes from the trap at the critical rotating speed: when the drag force is greater than the trapping force. With experimental verifications, the method can easily be employed to differentiate cells in terms of trapping forces due to different refractive indices. The proposed method is a simple, robust, accurate and noninvasive label-free technique for cell detection. 相似文献
17.
Magnetic micromanipulation using magnetic tweezers is a versatile biophysical technique and has been used for single-molecule unfolding, rheology measurements, and studies of force-regulated processes in living cells. This article describes an inexpensive magnetic tweezer setup for the application of precisely controlled forces up to 100 nN onto 5 microm magnetic beads. High precision of the force is achieved by a parametric force calibration method together with a real-time control of the magnetic tweezer position and current. High forces are achieved by bead-magnet distances of only a few micrometers. Applying such high forces can be used to characterize the local viscoelasticity of soft materials in the nonlinear regime, or to study force-regulated processes and mechanochemical signal transduction in living cells. The setup can be easily adapted to any inverted microscope. 相似文献
18.
光阱位置操纵系统的研究 总被引:4,自引:1,他引:3
利用He-Ne激光器,在LEICADMIRBE倒置显微镜上建立了可对生物样品进行三维操纵的光镊系统。在光镊的研制过程中,我们重点对光阱的横向位置操纵系统进行了研究。理论分析及实验表明,利用平移反射镜及转动反射镜的方案均可实现光阱位置的横向操纵。本文对平移反射镜及转动反射镜实现光阱位置操纵的机理进行了详细的论述,并对操纵过程中保证光阱质量不发生变化的条件进行了理论分析和说明。此外,比较分析表明,与平移反射镜系统相比,转镜系统在光镊技术中的应用可加快光镊操纵速度、简化光阱位置操纵系统的复杂程度、有利于系统开发成本的减少及开发周期的缩短。 相似文献
19.
We present here the principle of soft magnetic tweezers which improve the traditional magnetic tweezers allowing the simultaneous application and measurement of an arbitrary torque to a deoxyribonucleic acid (DNA) molecule. They take advantage of a nonlinear coupling regime that appears when a fast rotating magnetic field is applied to a superparamagnetic bead immersed in a viscous fluid. In this work, we present the development of the technique and we compare it with other techniques capable of measuring the torque applied to the DNA molecule. In this proof of principle, we use standard electromagnets to achieve our experiments. Despite technical difficulties related to the present implementation of these electromagnets, the agreement of measurements with previous experiments is remarkable. Finally, we propose a simple way to modify the experimental design of electromagnets that should bring the performances of the device to a competitive level. 相似文献
20.
M.I. SNIJDER-VAN AS B. RIEGER† B. JOOSTEN‡ V. SUBRAMANIAM C.G. FIGDOR‡ & J.S. KANGER 《Journal of microscopy》2009,233(1):84-92
The dynamics of cell surface membrane proteins plays an important role in cell–cell interactions. The onset of the interaction is typically not precisely controlled by current techniques, making especially difficult the visualization of early-stage dynamics. We have developed a novel method where optical tweezers are used to trap cells and precisely control in space and time the initiation of interactions between a cell and a functionalized surface. This approach is combined with total internal reflection fluorescence microscopy to monitor dynamics of membrane bound proteins. We demonstrate an accuracy of ∼2 s in determining the onset of the interaction. Furthermore, we developed a data analysis method to determine the dynamics of cell adhesion and the organization of membrane molecules at the contact area. We demonstrate and validate this approach by studying the dynamics of the green fluorescent protein tagged membrane protein activated leukocyte cell adhesion molecule expressed in K562 cells upon interaction with its ligand CD6 immobilized on a coated substrate. The measured cell spreading is in excellent agreement with existing theoretical models. Active redistribution of activated leukocyte cell adhesion molecule is observed from a clustered to a more homogenous distribution upon contact initiation. This redistribution follows exponential decay behaviour with a characteristic time of 35 s. 相似文献