Optical Trapping Force Research Articles

Cell manipulation by use of diamond microparticles as handles of optical tweezers

We report the experimental results of our using irregularly shaped diamond microparticles as handles for laser tweezers. Because of their irregular optical shape, control of the rotation of diamond microparticles can easily be achieved in a gradient force optical trap by use of a fixed linearly polarized beam with a fundamental Gaussian mode. By changing the laser focal plane upon a diamond particle near the liquid surface or interfaces, one can fully manipulate both the direction and speed of the rotation. The ability to manipulate a diamond-particle-tagged biological specimen by optical tweezers is discussed. The application of these particles as handles for optical tweezers is demonstrated by optical manipulation of biological cells. Independent movement of linear translation and rotation, with controllable rotation directions and speeds, is successfully achieved.

Effects of Spherical Aberration on Optical Trapping Forces forRayleigh Particles

The trapping force on Rayleigh particles in an optical tweezers system withan oil immersion objective is calculated by an electromagnetic model.The results indicate that the stability of particles trapped will beaffected by spherical aberration, which is caused by refractivedifference between objective oil and water solution, when the specimenmanipulated is suspended in a water solution. The trapping force anddepth of potential well will decrease and the minimum of laser power forensuring the stability of particles trapped will increase with theenhanced trapping depth.

Compact optical trapping microscope using a diode laser

Single beam gradient force optical trapping (laser tweezers) isincreasing in importance as a technique for microparticle and cellmanipulation and for the measurement of piconewton forces in aqueous media. Anoptical trap constructed using an optical microscope and a diode laser isdescribed here. The system incorporates novel beam steering optics allowingthe positioning of the trapping spot within the field of view of themicroscope. Despite its full functionality the system is compact, portable,relatively inexpensive and easy to construct and operate. A new scheme fortrapping force calibration is also reported and measurements compared totheoretical results. The trapping efficiency is found to be similar to thatobtained when using other trapping systems.

Changes in Hechtian strands in cold-hardened cells measured by optical microsurgery.

Optical microsurgical techniques were employed to investigate the mechanical properties of Hechtian strands in tobacco (Nicotiana tabacum) and Ginkgo biloba callus cells. Using optical tweezers, a 1. 5-microm diameter microsphere coated with concanavalin A was inserted though an ablated hole in the cell wall of a plasmolyzed cell and attached to a Hechtian strand. By displacing the adhered microsphere from equilibrium using the optical trapping force, the tensions of individual strands were determined. Measurements were made using both normal and cold-hardened cells, and in both cases, tensions were on the order of 10(-12) N. Significant differences were found in the binding strengths of cold-hardened and normal cultured cells. An increased number density of strands in cold-hardened G. biloba compared with normal cultured cells was also observed. Although no Hechtian strands were detected in any Arabidopsis callus cells, strands were present in leaf epidermal cells. Finally, the movement of attached microspheres was monitored along the outside of a strand while cycling the osmotic pressure.

Multiphoton fluorescence excitation in continuous-wave infrared optical traps

The multiphoton fluorescence excitation observed in acontinuous-wave (cw) single-beam gradient force optical trap(optical tweezers) is reported for latex beads labeled with ayellow-green fluorescent dye (BODIPY). The fluorescenceemission spectra of the yellow-green beads trapped and excited by thesame 1064-nm laser light are identical to the spectra excited by the365-nm UV light. The influence of the numerical aperture of theobjective on the slope of the log-log power-dependence has beendemonstrated for BODIPY-Oil solution under cw and pulsed-laserconditions. The possibility that three-photon excitation processoccurs is discussed within the context of a dog-bone saturationmodel. Other possibilities for the observed fluorescence excitationhave been discussed.

Theoretical investigation of the optical trapping force and torque on cylindrical micro-objects

We investigate theoretically the experimentally observed nature of the optical trapping force present on elongated, cylindrically shaped micro-objects. The objects chosen have either flat or spherical ends and elongated cylindrical bodies of various length-to-radii ratios. The trapping force, if present, occurs when the objects are placed in the focal region of a highly divergent laser beam. Results indicate that cylindrical objects can be trapped aligned with the laser beam axis.

Molecular Assembling by the Radiation Pressure of a Focused Laser Beam: Poly(N-isopropylacrylamide) in Aqueous Solution

Phase transitions in aqueous solutions of poly(N-isopropylacrylamide) (PNIPAM) with a molecular weight (M̄w) of 63 000 were achieved by irradiating the solutions (0.2−3.6 wt %) with an IR laser beam (1064 nm) through an optical microscope. First, a microparticle with the size of the focused laser beam was formed (≅1.5 μm). This microparticle continuously grew and after prolonged irradiation (up to 10 min), a microparticle with a maximum size of 25 μm was obtained. Upon further irradiation, the microparticle became unstable and finally disappeared. The importance of the optical alignment of the microscope/laser system is discussed. Particle formation was also found in D2O solutions of PNIPAM. These experimental results indicate that, besides a photothermal effect (heating up of the solution due to absorption of water at 1064 nm), there is influence of the “radiation force” upon particle formation and conformation properties of the polymer. The observations mentioned above are discussed in connection with the theory of the single beam gradient force optical trap for dielectric particles.

Applications of the optical trapping technique to analyze chemical reactions in single emulsion particles.

The applicability of the single beam gradient force optical trap and the optical levitation technique to investigate dynamic processes in microparticles is examined. The device allows to follow chemical reactions, like the emulsion polymerization and the ester hydrolysis reaction, in single emulsion droplets. Furthermore the usability of optical traps to investigate living cells is demonstrated.

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.

Two-photon fluorescence excitation in continuous-wave infrared optical tweezers

We report the observation of two-photon fluorescence excitation in a continuous-wave (cw) single-beam gradient force optical trap and demonstrate its use as an in situ probe to study the physiological state of an optically confined sample. In particular, a cw Nd:YAG (1064-nm) laser is used simultaneously to confine, and excite visible fluorescence from submicrometer regions of, cell specimens. Two-photon fluorescence emission spectra are presented for motile human sperm cells and immotile Chinese hamster ovary cells that have been labeled with nucleic acid (Propidium Iodide) and pH-sensitive (Snarf) fluorescent probes. The resulting spectra are correlated to light-induced changes in the physiological state experienced by the trapped cells. This spectral technique should prove extremely useful for monitoring cellular activity and the effects of confinement by optical tweezers.

Autofluorescence spectroscopy of optically trapped cells.

Cellular autofluorescence spectra were monitored in a single-beam gradient force optical trap ("optical tweezers") in order to probe the physiological effects of near infrared and UVA (320-400 nm) microirradiation. Prior to trapping, Chinese hamster ovary cells exhibited weak UVA-excited autofluorescence with maxima at 455 nm characteristic of beta-nicotinamide adenine dinucleotide (phosphate) emission. No strong effect of a 1064 nm NIR microbeam on fluorescence intensity and spectral characteristics was found during trapping, even for power densities up to 70 MW/cm2 and radiant exposures of 100 GJ/cm2. In contrast to the 1064 nm trap, a 760 nm trapping beam caused a two-fold autofluorescence increase within 5 min (about 20 GJ/cm2). Exposure to 365 nm UVA (1 W/cm2) during 1064 nm trapping significantly altered cellular autofluorescence, causing, within 10 min, a five-fold increase and a 6 nm red shift versus initial levels. We conclude that 1064 nm microbeams can be applied for an extended period without producing autofluorescence changes characteristic of alterations in the cellular redox state. However, 760 nm effects may occur via a two-photon absorption mechanism, which, in a manner similar to UVA exposure, alters the redox balance and places the cell in a state of oxidative stress.

A Differential Interference Contrast-Based Light Microscopic System for Laser Microsurgery and Optical Trapping of Selected Chromosomes during Mitosis In Vivo

Laser microsurgery and laser-generated optical force traps (optical tweezers) are both valuable light microscopic-based approaches for studying intra- and extracellular motility processes, including chromosome segregation during mitosis. Here we describe a system in use in our laboratory that allows living cells to be followed by high-resolution differential interference contrast (DIC) video-enhanced time-lapse light microscopy while selected mitotic organelles and spindle components are subjected to laser microsurgery and/or manipulation with an optical force trap. This system couples the output from two different Neodymium-YAG lasers to the same inverted light microscope equipped with both phase-contrast and de Senarmont compensation DIC optics, a motorized stage, and a high-resolution low-light-level CCD camera. Unlike similar systems using phase-contrast optics, our DIC-based system can image living cells in thin optical sections without contamination due to phase halos or out-of-focus object information. These advantages greatly facilitate laser-based light microscopic studies on mitotic organelles and components, including spindle poles (centrosomes) and kinetochores, which are at or below the resolution limit of the light microscope and buried within a large complex structure. When used in conjunction with image processing and high-resolution object-tracking techniques, our system provides new information on the roles that kinetochores and spindle microtubules play during chromosome segregation in plant and animal cells.

Calibration of light forces in optical tweezers

Axial and lateral optical-trapping forces on polystyrene and glass microbeads are measured as a function of sphere size and axial trapping position inside a specimen chamber containing water. A strong decrease of the light forces with increasing distance of the trapping position from the coverslip of the chamber is found. It is shown that beyond a certain maximal distance the trapping efficiency decreases substantially but trapping becomes possible in different, axial positions. We consider these effects to be accounted for by spherical aberration of the focused laser beam.

Buckling of a single microtubule by optical trapping forces: direct measurement of microtubule rigidity.

As major determinants of cell shape and polarity, microtubules are required to have suitable rigidity. However, our knowledge of the mechanical properties of microtubules is far from satisfactory. We report here a new method of measuring the flexural rigidity of a single microtubule by direct buckling using the optical trapping technique. Microtubule buckling was induced by applying a small longitudinal compressing force through an optically trapped microsphere that was firmly attached to the microtubule. Three ways of estimating the flexural rigidity of a continuous slender rod, one from the observed critical load of buckling and two from deflected lengths and angles of bending, yielded values which agreed well when applied to the analysis of buckling microtubules. Unexpectedly, we found that the rigidity was not constant as expected but was dependent on microtubule length. This length dependency explains the discrepancies among reported values of microtubule flexural rigidity measured by different methods. Comparing microtubules of identical lengths, microtubules assembled with brain-derived associated proteins (4 x 10(-23) Nm2 at around 10 microns in length) were four times more rigid than those assembled from purified tubulin and stabilized with taxol (1 x 10(-23) Nm2).

Parametric study of the forces on microspheres held by optical tweezers

Optical-trapping forces exerted on polystyrene microspheres are predicted and measured as a function of sphere size, laser spot size, and laser beam polarization. Axial and transverse forces are in good and excellent agreement, respectively, with a ray-optics model when the sphere diameter is ≥ 10 µm. Results are compared with results from an electromagnetic model when the sphere size is ≤ 1 µm. Axial trapping performance is found to be optimum when the numerical aperture of the objective lens is as large as possible, and when the trapped sphere is located just below the chamber cover slip. Forces in the transverse direction are not as sensitive to parametric variations as are the axial forces. These results are important as a first-order approximation to the forces that can be applied either directly to biological objects or by means of microsphere handles attached to the biological specimen.

Directed positioning of micronuclei in Paramecium tetraurelia with laser tweezers: absence of detectible damage after manipulation.

Possible covert damage from the use of the laser optical force trap (laser tweezers) to reposition micronuclei in Paramecium tetraurelia was assessed by measuring proliferation rates and postautogamous survival and mutation rates of cells after laser manipulations. No differences in subsequent daily proliferation rates among laser manipulated and various control classes of cells were seen. Similarly, the rates of postautogamous lethality and of "slow growth mutations" after repositioning of both micronuclei were not different from such rates in unmanipulated controls. In spite of extensive manipulations of micronuclei by the laser tweezers, there is no evidence of any damage induced by these manipulations. The laser tweezers therefore appears to be a tool of benign effect upon living cells, with tremendous potential use in many cell and developmental biological investigations.

Force of Single Kinesin Molecules Measured with Optical Tweezers

Isometric forces generated by single molecules of the mechanochemical enzyme kinesin were measured with a laser-induced, single-beam optical gradient trap, also known as optical tweezers. For the microspheres used in this study, the optical tweezers was spring-like for a radius of 100 nanometers and had a maximum force region at a radius of approximately 150 nanometers. With the use of biotinylated microtubules and special streptavidin-coated latex microspheres as handles, microtubule translocation by single squid kinesin molecules was reversibly stalled. The stalled microtubules escaped optical trapping forces of 1.9 +/- 0.4 piconewtons. The ability to measure force parameters of single macromolecules now allows direct testing of molecular models for contractility.

Prediction and measurement of the optical trapping forces on a microscopic dielectric sphere

Prediction and measurement of the optical trapping forces on a microscopic dielectric sphere

Optical trapping in animal and fungal cells using a tunable, near-infrared titanium-sapphire laser

We have compared two different laser-induced optical light traps for their utility in moving organelles within living animal cells and walled fungal cells. The first trap employed a continuous wave neodymium-yttrium aluminum garnet (Nd-YAG) laser at a wave-length of 1.06 μm. A second trap was constructed using a titanium-sapphire laser tunable from 700 to 1000 nm. With the latter trap we were able to achieve much stronger traps with less laser power and without damage to either mitochondria or spindles. Chromosomes and nuclei were easily displaced, nucleoli were separated and moved far away from interphase nuclei, and Woronin bodies were removed from septa. In comparison, these manipulations were not possible with the Nd-YAG laser-induced trap. The optical force trap induced by the tunable titanium-sapphire laser should find wide application in experimental cell biology because the wavelength can be selected for maximization of force production and minimization of energy absorption which leads to unwanted cell damage.

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

AbstractWe 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.