Laser induced cell fusion in combination with optical tweezers: the laser cell fusion trap.

A single-beam gradient force optical trap was combined with a pulsed UV laser microbeam in order to perform laser induced cell fusion. This combination offers the possibility to selectively fuse two single cells without critical chemical or electrical treatment. The optical trap was created by directing a Nd:YAG laser, at a wavelength of 1.06 microns, into a microscope and focusing the laser beam with a high numerical aperture objective. The UV laser microbeam, produced by a nitrogen-pumped dye laser (366 nm), was collinear with the trapping beam. Once inside the trap, two cells could be fused with several pulses of the UV laser microbeam, attenuated to an energy of approximately 1 microJ/pulse in the object plane. This method of laser induced cell fusion should provide increased selectivity and efficiency in generating viable hybrid cells.     

Detection of forces and displacements along the axial direction in an optical trap

We present measurements of the forces on, and displacements of, an optically trapped bead along the propagation direction of the trapping laser beam (the axial direction). In a typical experimental configuration, the bead is trapped in an aqueous solution using an oil-immersion, high-numerical-aperture objective. This refractive index mismatch complicates axial calibrations due to both a shift of the trap center along the axial direction and spherical aberrations. In this work, a known DNA template was unzipped along the axial direction and its characteristic unzipping force-extension data were used to determine 1), the location of the trap center along the axial direction; 2), the axial displacement of the bead from the trap center; and 3), the axial force exerted on the bead. These axial calibrations were obtained for trap center locations up to approximately 4 microm into the aqueous solution and with axial bead displacements up to approximately 600 nm from the trap center. In particular, the axial trap stiffness decreased substantially when the trap was located further into the aqueous solution. This approach, together with conventional lateral calibrations, results in a more versatile optical trapping instrument that is accurately calibrated in all three dimensions.     

Long-Distance Axial Trapping with Focused Annular Laser Beams

Focusing an annular laser beam can improve the axial trapping efficiency due to the reduction of the scattering force, which enables the use of a lower numerical aperture (NA) objective lens with a long working distance to trap particles in deeper aqueous medium. In this paper, we present an axicon-to-axicon scheme for producing parallel annular beams with the advantages of higher efficiency compared with the obstructed beam approach. The validity of the scheme is verified by the observation of a stable trapping of silica microspheres with relatively low NA microscope objective lenses (NA = 0.6 and 0.45), and the axial trapping depth of 5 mm is demonstrated in experiment.     

Optical Trapping of Nanoparticles

Optical trapping is a technique for immobilizing and manipulating small objects in a gentle way using light, and it has been widely applied in trapping and manipulating small biological particles. Ashkin and co-workers first demonstrated optical tweezers using a single focused beam¹. The single beam trap can be described accurately using the perturbative gradient force formulation in the case of small Rayleigh regime particles¹. In the perturbative regime, the optical power required for trapping a particle scales as the inverse fourth power of the particle size. High optical powers can damage dielectric particles and cause heating. For instance, trapped latex spheres of 109 nm in diameter were destroyed by a 15 mW beam in 25 sec¹, which has serious implications for biological matter^2,3.A self-induced back-action (SIBA) optical trapping was proposed to trap 50 nm polystyrene spheres in the non-perturbative regime⁴. In a non-perturbative regime, even a small particle with little permittivity contrast to the background can influence significantly the ambient electromagnetic field and induce a large optical force. As a particle enters an illuminated aperture, light transmission increases dramatically because of dielectric loading. If the particle attempts to leave the aperture, decreased transmission causes a change in momentum outwards from the hole and, by Newton''s Third Law, results in a force on the particle inwards into the hole, trapping the particle. The light transmission can be monitored; hence, the trap can become a sensor. The SIBA trapping technique can be further improved by using a double-nanohole structure.The double-nanohole structure has been shown to give a strong local field enhancement^5,6. Between the two sharp tips of the double-nanohole, a small particle can cause a large change in optical transmission, thereby inducing a large optical force. As a result, smaller nanoparticles can be trapped, such as 12 nm silicate spheres⁷ and 3.4 nm hydrodynamic radius bovine serum albumin proteins⁸. In this work, the experimental configuration used for nanoparticle trapping is outlined. First, we detail the assembly of the trapping setup which is based on a Thorlabs Optical Tweezer Kit. Next, we explain the nanofabrication procedure of the double-nanohole in a metal film, the fabrication of the microfluidic chamber and the sample preparation. Finally, we detail the data acquisition procedure and provide typical results for trapping 20 nm polystyrene nanospheres.     

Forces of a single-beam gradient laser trap on a dielectric sphere in the ray optics regime

We calculate the forces of single-beam gradient radiation pressure laser traps, also called “optical tweezers,” on micron-sized dielectric spheres in the ray optics regime. This serves as a simple model system for describing laser trapping and manipulation of living cells and organelles within cells. The gradient and scattering forces are defined for beams of complex shape in the ray-optics limit. Forces are calculated over the entire cross-section of the sphere using TEM₀₀ and TEM₀₁^* mode input intensity profiles and spheres of varying index of refraction. Strong uniform traps are possible with force variations less than a factor of 2 over the sphere cross-section. For a laser power of 10 mW and a relative index of refraction of 1.2 we compute trapping forces as high as ~ 1.2 × 10^-6 dynes in the weakest (backward) direction of the gradient trap. It is shown that good trapping requires high convergence beams from a high numerical aperture objective. A comparison is given of traps made using bright field or differential interference contrast optics and phase contrast optics.     

Mechanical force characterization in manipulating live cells with optical tweezers

Laser trapping with optical tweezers is a noninvasive manipulation technique and has received increasing attentions in biological applications. Understanding forces exerted on live cells is essential to cell biomechanical characterizations. Traditional numerical or experimental force measurement assumes live cells as ideal objects, ignoring their complicated inner structures and rough membranes. In this paper, we propose a new experimental method to calibrate the trapping and drag forces acted on live cells. Binding a micro polystyrene sphere to a live cell and moving the mixture with optical tweezers, we can obtain the drag force on the cell by subtracting the drag force on the sphere from the total drag force on the mixture, under the condition of extremely low Reynolds number. The trapping force on the cell is then obtained from the drag force when the cell is in force equilibrium state. Experiments on numerous live cells demonstrate the effectiveness of the proposed force calibration approach.     

Axial Optical Traps: A New Direction for Optical Tweezers

Optical tweezers have revolutionized our understanding of the microscopic world. Axial optical tweezers, which apply force to a surface-tethered molecule by directly moving either the trap or the stage along the laser beam axis, offer several potential benefits when studying a range of novel biophysical phenomena. This geometry, although it is conceptually straightforward, suffers from aberrations that result in variation of the trap stiffness when the distance between the microscope coverslip and the trap focus is being changed. Many standard techniques, such as back-focal-plane interferometry, are difficult to employ in this geometry due to back-scattered light between the bead and the coverslip, whereas the noise inherent in a surface-tethered assay can severely limit the resolution of an experiment. Because of these complications, precision force spectroscopy measurements have adapted alternative geometries such as the highly successful dumbbell traps. In recent years, however, most of the difficulties inherent in constructing a precision axial optical tweezers have been solved. This review article aims to inform the reader about recent progress in axial optical trapping, as well as the potential for these devices to perform innovative biophysical measurements.     

Fabrication and Operation of a Nano-Optical Conveyor Belt

The technique of using focused laser beams to trap and exert forces on small particles has enabled many pivotal discoveries in the nanoscale biological and physical sciences over the past few decades. The progress made in this field invites further study of even smaller systems and at a larger scale, with tools that could be distributed more easily and made more widely available. Unfortunately, the fundamental laws of diffraction limit the minimum size of the focal spot of a laser beam, which makes particles smaller than a half-wavelength in diameter hard to trap and generally prevents an operator from discriminating between particles which are closer together than one half-wavelength. This precludes the optical manipulation of many closely-spaced nanoparticles and limits the resolution of optical-mechanical systems. Furthermore, manipulation using focused beams requires beam-forming or steering optics, which can be very bulky and expensive. To address these limitations in the system scalability of conventional optical trapping our lab has devised an alternative technique which utilizes near-field optics to move particles across a chip. Instead of focusing laser beams in the far-field, the optical near field of plasmonic resonators produces the necessary local optical intensity enhancement to overcome the restrictions of diffraction and manipulate particles at higher resolution. Closely-spaced resonators produce strong optical traps which can be addressed to mediate the hand-off of particles from one to the next in a conveyor-belt-like fashion. Here, we describe how to design and produce a conveyor belt using a gold surface patterned with plasmonic C-shaped resonators and how to operate it with polarized laser light to achieve super-resolution nanoparticle manipulation and transport. The nano-optical conveyor belt chip can be produced using lithography techniques and easily packaged and distributed.     

Enhancing the Strength of an Optical Trap by Truncation

Optical traps (tweezers) are beginning to be used with increasing efficacy in diverse studies in the biological and biomedical sciences. We report here results of a systematic study aimed at enhancing the efficiency with which dielectric (transparent) materials can be optically trapped. Specifically, we investigate how truncation of the incident laser beam affects the strength of an optical trap in the presence of a circular aperture. Apertures of various sizes have been used by us to alter the beam radius, thereby changing the effective numerical aperture and intensity profile. We observe significant enhancement of the radial and axial trap stiffness when an aperture is used to truncate the beam compared to when no aperture was used, keeping incident laser power constant. Enhancement in trap stiffness persists even when the beam intensity profile is modulated. The possibility of applying truncation to multiple traps is explored; to this end a wire mesh is utilized to produce multiple trapping that also alters the effective numerical aperture. The use of a mesh leads to reduction in trap stiffness compared to the case when no wire mesh is used. Our findings lead to a simple-to-implement and inexpensive method of significantly enhancing optical trapping efficiency under a wide range of circumstances.     

Properties and parameters of the electron beam injected into the mirror magnetic trap of a plasma accelerator

The parameters of the injector of an axial plasma beam injected into a plasma accelerator operating on the basis of gyroresonance acceleration of electrons in the reverse magnetic field are determined. The trapping of the beam electrons into the regime of gyroresonance acceleration is numerically simulated by the particle- in-cell method. The optimal time of axial injection of the beam into a magnetic mirror trap is determined. The beam parameters satisfying the condition of efficient particle trapping into the gyromagnetic autoresonance regime are found.     

Euler buckling-induced folding and rotation of red blood cells in an optical trap

We investigate the physics of an optically driven micromotor of biological origin. When a single, live red blood cell (RBC) is placed in an optical trap, the normal biconcave disc shape of the cell is observed to fold into a rod-like shape. If the trapping laser beam is circularly polarized, the folded RBC rotates. A model based on geometric considerations, using the concept of buckling instabilities, captures the folding phenomenon; the rotation of the cell is rationalized using the Poincaré sphere. Our model predicts that (i) at a critical power of the trapping laser beam the RBC shape undergoes large fluctuations, and (ii) the torque that is generated is proportional to the power of the laser beam. These predictions are verified experimentally. We suggest a possible mechanism for the emergence of birefringent properties in the RBC in the folded state.     

An integrated instrumental setup for the combination of atomic force microscopy with optical spectroscopy

In recent years, the study of single biomolecules using fluorescence microscopy and atomic force microscopy (AFM) techniques has resulted in a plethora of new information regarding the physics underlying these complex biological systems. It is especially advantageous to be able to measure the optical, topographical, and mechanical properties of single molecules simultaneously. Here an AFM is used that is especially designed for integration with an inverted optical microscope and that has a near-infrared light source (850 nm) to eliminate interference between the optical experiment and the AFM operation. The Tip Assisted Optics (TAO) system consists of an additional 100 x 100-microm(2) X-Y scanner for the sample, which can be independently and simultaneously used with the AFM scanner. This allows the offset to be removed between the confocal optical image obtained with the sample scanner and the simultaneously acquired AFM topography image. The tip can be positioned exactly into the optical focus while the user can still navigate within the AFM image for imaging or manipulation of the sample. Thus the tip-enhancement effect can be maximized and it becomes possible to perform single molecule manipulation experiments within the focus of a confocal optical image. Here this is applied to simultaneous measurement of single quantum dot fluorescence and topography with high spatial resolution.     

An integrated laser trap/flow control video microscope for the study of single biomolecules

We have developed an integrated laser trap/flow control video microscope for mechanical manipulation of single biopolymers. The instrument is automated to maximize experimental throughput. A single-beam optical trap capable of trapping micron-scale polystyrene beads in the middle of a 200-microm-deep microchamber is used, making it possible to insert a micropipette inside this chamber to hold a second bead by suction. Together, these beads function as easily exchangeable surfaces between which macromolecules of interest can be attached. A computer-controlled flow system is used to exchange the liquid in the chamber and to establish a flow rate with high precision. The flow and the optical trap can be used to exert forces on the beads, the displacements of which can be measured either by video microscopy or by laser deflection. To test the performance of this instrument, individual biotinylated DNA molecules were assembled between two streptavidin beads, and the DNA elasticity was characterized using both laser trap and flow forces. DNA extension under varying forces was measured by video microscopy. The combination of the flow system and video microscopy is a versatile design that is particularly useful for the study of systems susceptible to laser-induced damage. This capability was demonstrated by following the translocation of transcribing RNA polymerase up to 650 s.     

Single-molecule manipulation of macromolecules on GUV or SUV membranes using optical tweezers

Despite their wide applications in soluble macromolecules, optical tweezers have rarely been used to characterize the dynamics of membrane proteins, mainly due to the lack of model membranes compatible with optical trapping. Here, we examined optical trapping and mechanical properties of two potential model membranes, giant and small unilamellar vesicles (GUVs and SUVs, respectively) for studies of membrane protein dynamics. We found that optical tweezers can stably trap GUVs containing iodixanol with controlled membrane tension. The trapped GUVs with high membrane tension can serve as a force sensor to accurately detect reversible folding of a DNA hairpin or membrane binding of synaptotagmin-1 C2AB domain attached to the GUV. We also observed that SUVs are rigid enough to resist large pulling forces and are suitable for detecting protein conformational changes induced by force. Our methodologies may facilitate single-molecule manipulation studies of membrane proteins using optical tweezers.     

Targeted cell immobilization by ultrasound microbeam

Various techniques exerting mechanical stress on cells have been developed to investigate cellular responses to externally controlled stimuli. Fundamental mechanotransduction processes about how applied physical forces are converted into biochemical signals have often been examined by transmitting such forces through cells and probing its pathway at cellular levels. In fact, many cellular biomechanics studies have been performed by trapping (or immobilizing) individual cells, either attached to solid substrates or suspended in liquid media. In that context, we demonstrated two‐dimensional acoustic trapping, where a lipid droplet of 125 µm in diameter was directed transversely toward the focus (or the trap center) similar to that of optical tweezers. Under the influence of restoring forces created by a 30 MHz focused ultrasound beam, the trapped droplet behaved as if tethered to the focus by a linear spring. In order to apply this method to cellular manipulation in the Mie regime (cell diameter > wavelength), the availability of sound beams with its beamwidth approaching cell size is crucial. This can only be achieved at a frequency higher than 100 MHz. We define ultrasound beams in the frequency range from 100 MHz to a few GHz as ultrasound microbeams because the lateral beamwidth at the focus would be in the micron range. Hence a zinc oxide (ZnO) transducer that was designed and fabricated to transmit a 200 MHz focused sound beam was employed to immobilize a 10 µm human leukemia cell (K‐562) within the trap. The cell was laterally displaced with respect to the trap center by mechanically translating the transducer over the focal plane. Both lateral displacement and position trajectory of the trapped cell were probed in a two‐dimensional space, indicating that the retracting motion of these cells was similar to that of the lipid droplets at 30 MHz. The potential of this tool for studying cellular adhesion between white blood cells and endothelial cells was discussed, suggesting its capability as a single cell manipulator. Biotechnol. Bioeng. 2011; 108:1643–1650. © 2011 Wiley Periodicals, Inc.     

Optical tweezers for the micromanipulation of plant cytoplasm and organelles

Laser trapping of micron-sized particles can be achieved utilizing the radiation pressure generated by a focused infrared laser beam. Thus, it is theoretically possible to trap and manipulate organelles within the cytoplasm and remodel the architecture of the cytoplasm and membrane systems. Here we describe recent progress, using this under utilized technology, in the manipulation of cytoplasmic strands and organelles in plant cells.     

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.Download video file.^{(62M, mov)}     

Low‐photobleaching line‐scanning confocal microscopy using dual inclined beams

Confocal microscopy is an indispensable tool for biological imaging due to its high resolution and optical sectioning capability. However, its slow imaging speed and severe photobleaching have largely prevented further applications. Here, we present dual inclined beam line‐scanning (LS) confocal microscopy. The reduced excitation intensity of our imaging method enabled a 2‐fold longer observation time of fluorescence compared to traditional LS microscopy while maintaining a good sectioning capability and single‐molecule sensitivity. We characterized the performance of our method and applied it to subcellular imaging and three‐dimensional single‐molecule RNA imaging in mammalian cells.      

Optical Manipulation of Dielectric Nanoparticles with Au Micro-racetrack Resonator by Constructive Interference of Surface Plasmon Waves

We design a gold micro-racetrack resonator (Au-MRR) which can tightly trap and drive the dielectric nanoparticle to rotate around the circuit of racetrack with an adjustable velocity. Since the surface plasmon waves can be excited and obey the resonance condition of the Au-MRR, the optics force can be strengthened observably due to the resonance. The optical forces applied on dielectric nanoparticle are discussed by utilizing the Maxwell’s stress tensor integration with a numerical finite element method. The depth of longitudinal trapping potential well in the Au-MRR is four times as large as that of a straight waveguide. At the same level of input power, the velocity of particle with radius of 50 nm driven by optical forces on Au-MRR is 200 times larger than that on a straight waveguide. Further, we explore the motion behavior of single nanoparticle lies on different position of Au-MRR, which can provide the details to trap and manipulate multiple nanoparticles and predict their trace of movement. This optimum geometry of Au-MRR allows further enhancement of the optical forces which is expected to realize all-optical on-chip manipulation of nanoparticles, biomolecules, and many other nanomanipulation applications.     

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).¹ However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.² However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.³ A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET''s have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.^4,5This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.^6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP''s generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.^9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.¹¹ Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.¹⁰ The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.Download video file.^{(57M, mov)}