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.     

Wavelength dependence of cell cloning efficiency after optical trapping.

A study on clonal growth in Chinese hamster ovary (CHO) cells was conducted after exposure to optical trapping wavelengths using Nd:YAG (1064 nm) and tunable titanium-sapphire (700-990 nm) laser microbeam optical traps. The nuclei of cells were exposed to optical trapping forces at various wavelengths, power densities, and durations of exposure. Clonal growth generally decreased as the power density and the duration of laser exposure increased. A wavelength dependence of clonal growth was observed, with maximum clonability at 950-990 nm and least clonability at 740-760 nm and 900 nm. Moreover, the most commonly used trapping wavelength, 1064 nm from the Nd:YAG laser, strongly reduced clonability, depending upon the power density and exposure time. The present study demonstrates that a variety of optical parameters must be considered when applying optical traps to the study of biological problems, especially when survival and viability are important factors. The ability of the optical trap to alter either the structure or biochemistry of the process being probed with the trapping beam must be seriously considered when interpreting experimental results.     

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.     

Single beam optical trapping integrated in a confocal microscope for biological applications.

Confocal microscopy is very useful in biology because of its three dimensional imaging capacities and has proven to be an excellent tool to study the 3D organization of, for instance, cell structures. This property of confocal microscopy makes it also very suitable for observation during guidance of the three dimensional manipulation of single cells or cell elements. Therefore we decided to integrate a confocal microscope and a single beam optical manipulator into a single instrument. The advantage of optical manipulation over mechanical techniques is that it is non-invasive and therefore may be applied on living (micro-) organisms and cells. The creation of an effective single beam optical trap requires the use of a high numerical aperture (N.A.) objective to focus the laser beam. In this paper we briefly discuss the vertical or axial force exerted on a sphere in a single beam trap. The axial force on a sphere placed on the optical axis, caused by reflection and refraction, is calculated applying a electromagnetic vector diffraction theory to determine the field distribution in the focal region. One of the results is that the particle also experiences a vertical trapping force towards the focusing lens when it is in the strongly convergent part of the field in addition to the known negative signed trapping force in the divergent part of the field. Further we describe an instrumental approach to realize optical trapping in which the optical trap position is controlled by moving the focusing objective only.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Experimental and theoretical investigations on the validity of the geometrical optics model for calculating the stability of optical traps.

We have developed a computer program based on the geometrical optics approach proposed by Roosen to calculate the forces on dielectric spheres in focused laser beams. We have explicitly taken into account the polarization of the laser light and thd divergence of the laser beam. The model can be used to evaluate the stability of optical traps in a variety of different optical configurations. Our calculations explain the experimental observation by Ashkin that a stable single-beam optical trap, without the help of the gravitation force, can be obtained with a strongly divergent laser beam. Our calculations also predict a different trap stability in the directions orthogonal and parallel to the polarization direction of the incident light. Different experimental methods were used to test the predictions of the model for the gravity trap. A new method for measuring the radiation force along the beam axis in both the stable and instable regions is presented. Measurements of the radiation force on polystyrene spheres with diameters of 7.5 and 32 microns in a TEM00-mode laser beam showed a good qualitative correlation with the predictions and a slight quantitative difference. The validity of the geometrical approximations involved in the model will be discussed for spheres of different sizes and refractive indices.     

Plasmonic Optical Trapping in Biologically Relevant Media

We present plasmonic optical trapping of micron-sized particles in biologically relevant buffer media with varying ionic strength. The media consist of 3 cell-growth solutions and 2 buffers and are specifically chosen due to their widespread use and applicability to breast-cancer and angiogenesis studies. High-precision rheological measurements on the buffer media reveal that, in all cases excluding the 8.0 pH Stain medium, the fluids exhibit Newtonian behavior, thereby enabling straightforward measurements of optical trap stiffness from power-spectral particle displacement data. Using stiffness as a trapping performance metric, we find that for all media under consideration the plasmonic nanotweezers generate optical forces 3–4x a conventional optical trap. Further, plasmonic trap stiffness values are comparable to those of an identical water-only system, indicating that the performance of a plasmonic nanotweezer is not degraded by the biological media. These results pave the way for future biological applications utilizing plasmonic optical traps.     

Behaviour of male European corn borer, Ostrinia nubilalis Hübner (Lep.; Crambidae) towards pheromone-baited delta traps, bucket traps and wire mesh cone traps

Abstract:  Pheromone-baited traps are used to monitor the flight of European corn borer (ECB; Ostrinia nubilalis ). Traps of various designs are available: bucket traps, delta sticky traps and wire mesh cone traps. However, these traps are not all equally efficient and little is known about the reasons for this difference in efficiency. We investigated the behaviour of ECB males towards bucket traps and delta traps by means of observation in a wind tunnel. We also carried out observations and capture trials with delta traps and wire mesh cone traps in field conditions. Our laboratory studies showed that ECB males were not optimally attracted and were poorly captured by a pheromone baited bucket trap. Furthermore, they were shown to readily enter delta traps in the wind tunnel but were caught after more than three passages through this trap. Field studies showed that wire mesh cone traps captured approximately six times more ECB males than delta traps. Observation of the behaviour of ECB males showed that this difference was due to more efficient moth capture rather than greater attraction of the moth. In total, 31.5% of the 219 males observed close to wire mesh cone traps were caught, vs. 2.5% of the 520 males observed close to delta traps. This greater efficiency is due to a better capture rate of the attracted males by wire mesh cone traps than by delta traps.     

Modified pheromone traps help reduce bycatch of bark‐beetle natural enemies

• 1 Thanasimus formicarius and Temnochila caerulea, two of the main predators of Ips sexdentatus, a well‐known forest pest in Southern Europe, are captured in high numbers when trapping I. sexdentatus as a result of the kairomonal effect of the lures used.
• 2 A preliminary field trial showed that predators could survive for at least 1 week within trapping containers, although predator mutilation and high predator death rates were observed.
• 3 Different modifications of conventional multiple funnel and slot traps with the objective of reducing natural enemy entrance into trap containers were bioassayed in field experiments conducted over four seasons. Based on the larger sizes of predators, different designs using welded wire‐mesh screens improved performance to different extents. Providing escape windows just above the screen on multiple funnel traps gave the most promising results, including when effect sizes among all tested designs were compared.
• 4 Thus, a simple modification of the lowest funnel of the multiple funnel traps would reduce the bycatch of T. formicarius and T. caerulea, hence improving the efficiency of trapping programmes by lowering the likely impact on natural populations of these predators.

     

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.     

Directed positioning of nuclei in living Paramecium tetraurelia: Use of the laser optical force trap for developmental biology

We have constructed a laser optical force trap (“laser tweezers”) by coupling an Nd:YAG laser to an optical microscope with a high numerical aperture objective. The laser beam (approximately 0.1 W power) is focused to a diffraction-limited spot at the specimen plane of the objective: the wavelength chosen (1,064 nm) is not strongly absorbed by most biological materials and is thus not ablative. Because the intensity of the laser beam increases towards the center of the focal spot, small particles brought near the spot will be attracted to the center and held there. Movement of the laser beam will tend to move any trapped particles with it. The laser tweezers can permit precise, nondestructive repositioning of small structures inside a living cell, without recourse to micromanipulators. Initial work has involved the use of laser tweezers on cells of Paramecium tet-raurelia held by a rotocompressor. We have been able to trap and reposition small organelles, especially the highly refractile structures known as crystals. Using a trapped crystal as a “tool”, we have been able to push micronuclei and other structures for many micrometers to virtually any desired location in a cell. In spite of extended exposure of specific structures and of individual cells to the laser beam, no damage has been detectible. Exposed cells, which were removed from the rotocompres-sor and cultured, showed complete viabilty. The laser tweezers technique shows tremendous potential for applications to the study of many fundamental cellular and developmental phenomena in paramecia and other ciliates. For example, we intend to use this technique to investigate temporal and spatial characteristics of nuclear determining regions during sexual reorganization in Paramecium. © 1992 Wiley-Liss, Inc.     

Label‐free differentiation of human immunodeficiency virus‐1 infected from uninfected cells using transmission measurement

Transmission measurement has been perceived as a potential candidate for label‐free investigation of biological material. It is a real‐time, label‐free and non‐invasive optical detection technique that has found wide applications in pharmaceutical industry as well as the biological and medical fields. Combining transmission measurement with optical trapping has emerged as a powerful tool allowing stable sample trapping, while also facilitating transmittance data analysis. In this study, a near‐infrared laser beam emitting at a wavelength of 1064 nm was used for both optical trapping and transmission measurement investigation of human immunodeficiency virus 1 (HIV‐1) infected and uninfected TZM‐bl cells. The measurements of the transmittance intensity of individual cells in solution were carried out using a home built optical trapping system combined with laser transmission setup using a single beam gradient trap. Transmittance spectral intensity patterns revealed significant differences between the HIV‐1 infected and uninfected cells. This result suggests that the transmittance data analysis technique used in this study has the potential to differentiate between infected and uninfected TZM‐bl cells without the use of labels. The results obtained in this study could pave a way into developing an HIV‐1 label‐free diagnostic tool with possible applications at the point of care .     

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.     

Characterization of photodamage to escherichia coli in optical traps

Optical tweezers (infrared laser-based optical traps) have emerged as a powerful tool in molecular and cell biology. However, their usefulness has been limited, particularly in vivo, by the potential for damage to specimens resulting from the trapping laser. Relatively little is known about the origin of this phenomenon. Here we employed a wavelength-tunable optical trap in which the microscope objective transmission was fully characterized throughout the near infrared, in conjunction with a sensitive, rotating bacterial cell assay. Single cells of Escherichia coli were tethered to a glass coverslip by means of a single flagellum: such cells rotate at rates proportional to their transmembrane proton potential (. J. Mol. Biol. 138:541-561). Monitoring the rotation rates of cells subjected to laser illumination permits a rapid and quantitative measure of their metabolic state. Employing this assay, we characterized photodamage throughout the near-infrared region favored for optical trapping (790-1064 nm). The action spectrum for photodamage exhibits minima at 830 and 970 nm, and maxima at 870 and 930 nm. Damage was reduced to background levels under anaerobic conditions, implicating oxygen in the photodamage pathway. The intensity dependence for photodamage was linear, supporting a single-photon process. These findings may help guide the selection of lasers and experimental protocols best suited for optical trapping work.     

Stabilizing the Central Part of Tropomyosin Increases the Bending Stiffness of the Thin Filament

A two-beam optical trap was used to measure the bending stiffness of F-actin and reconstructed thin filaments. A dumbbell was formed by a filament segment attached to two beads that were held in the two optical traps. One trap was static and held a bead used as a force transducer, whereas an acoustooptical deflector moved the beam holding the second bead, causing stretch of the dumbbell. The distance between the beads was measured using image analysis of micrographs. An exact solution to the problem of bending of an elastic filament attached to two beads and subjected to a stretch was used for data analysis. Substitution of noncanonical residues in the central part of tropomyosin with canonical ones, G126R and D137L, and especially their combination, caused an increase in the bending stiffness of the thin filaments. The data confirm that the effect of these mutations on the regulation of actin-myosin interactions may be caused by an increase in tropomyosin stiffness.     

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.     

Rapid 3D fluorescence imaging of individual optically trapped living immune cells

We demonstrate an approach to rapidly characterize living suspension cells in 4 dimensions while they are immobilized and manipulated within optical traps. A single, high numerical aperture objective lens is used to separate the imaging plane from the trapping plane. This facilitates full control over the position and orientation of multiple trapped cells using a spatial light modulator, including directed motion and object rotation, while also allowing rapid 4D imaging. This system is particularly useful in the handling and investigation of the behavior of non‐adherent immune cells. We demonstrate these capabilities by imaging and manipulating living, fluorescently stained Jurkat T cells. (© 2015 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Pheromone trapping of the sugarcane weevil borer, Rhabdoscelus obscurus (Boisduval) (Coleoptera: Curculionidae): an evaluation of trap design and placement in the field

Abstract  The sugarcane weevil borer ( Rhabdoscelus obscurus ) is a pest of sugarcane and palm plantations in high rainfall areas of far north Queensland. Pheromone mass trapping of adult borers is an effective method of monitoring and may also impact on their population densities. Trials to evaluate different designs of pheromone trap showed the 'water trap' to be the most effective in capturing the highest number of adult borers. The water trap is a 20 cm-diameter pot with a plastic bag inserted to hold water with pheromone lures and cane pieces held together in a plastic container suspended over the water from a square of wire mesh. Results also showed that adult borers in adjacent fields are able to sense the lures and migrate into treated fields. The sex ratio of captured borers in split-cane traps (which is a standard population monitoring tool) was male biased from February to April but was more balanced later in the year. However, the pheromone traps mainly attracted female borers all year round, except during May. Thus, we recommend pheromone traps to be deployed early in the season (November to early December) to attract the maximum number of adult females at a critical time for the population as it starts to build up. In addition, placing pheromone traps inside cane paddocks did not improve the total catch in most cases. Therefore, placing pheromone traps under the shade of trees outside the paddock or at the edge of the paddock under sugarcane can be a feasible mass trapping method that can easily be implemented by cane growers.     

Optical tweezers based force measurement system for quantitating binding interactions: system design and application for the study of bacterial adhesion

An optical force measurement system for quantitating forces in the pN range between micrometer-sized objects has been developed. The system was based upon optical tweezers in combination with a sensitive position detection system and constructed around an inverted microscope. A trapped particle in the focus of the high numerical aperture microscope-objective behaves like an omnidirectional mechanical spring in response to an external force. The particle's displacement from the equilibrium position is therefore a direct measure of the exerted force. A weak probe laser beam, focused directly below the trapping focus, was used for position detection of the trapped particle (a polystyrene bead). The bead and the condenser focus the light to a distinct spot in the far field, monitored by a position sensitive detector. Various calibration procedures were implemented in order to provide absolute force measurements. The system has been used to measure the binding forces between Escherichia coli bacterial adhesins and galabiose-functionalized beads.