This PDF file contains the front matter associated with SPIE Proceedings Volume 6644, including the Title Page, Copyright information, Table of Contents, Introduction (if any), and the Conference Committee listing.

Optical tweezers have become an important tool for the manipulation of single biomolecules. However, their application to stretching biopolymers is usually limited to molecules that are several microns in length because conventional optical tweezers manipulate molecules laterally in the focal plane of the microscope objective, a mode in which steric hindrances from the attached microsphere and the surface are substantial. In order to study the properties of short DNA fragments that are typically 1000 bp long, we used optical tweezers in the axial direction to pull microsphere away from the cover glass surface. The microsphere was held in the linear region of the optical potential where the optical force is least sensitive to the bead position. By varying the laser intensity, different stretching forces were applied to the DNA molecule, and the axial position of the tethered microsphere was obtained from its diffraction pattern. The results indicate that the wormlike chain model is still valid for such short DNA fragments.

A key step in the assembly of many viruses is the packaging of double-stranded DNA into a viral procapsid (an empty protein shell) by the action of an ATP-powered portal motor complex. We have developed methods to measure the packaging of single DNA molecules into single viral proheads in real time using optical tweezers. We can measure DNA binding and initiation of translocation, the DNA translocation dynamics, and the filling of the capsid against resisting forces. In addition to studying bacteriophage φ29, we have recently extended these methods to study the E. coli bacteriophages λ and T4, two important model systems in molecular biology. The three systems have different capsid sizes/shapes, genome lengths, and biochemical and structural differences in their packaging motors. Here, we compare and contrast these three systems. We find that all three motors translocate DNA processively and generate very large forces, each exceeding 50 piconewtons, ~20x higher force than generated by the skeletal muscle myosin 2 motor. This high force generation is required to overcome the forces resisting the confinement of the stiff, highly charged DNA at high density within the viral capsids. However, there are also striking differences between the three motors: they exhibit different DNA translocation rates, degrees of static and dynamic disorder, responses to load, and pausing and slipping dynamics.

Dual - beam optical tweezers experiments subject single molecules of DNA to high forces (~ 300 pN) with 0.1 pN accuracy, probing the energy and specificity of nucleic acid - ligand structures. Stretching phage λ-DNA reveals an increase in the applied force up to a critical force known as the overstretching transition. In this region, base pairing and stacking are disrupted as double stranded DNA (dsDNA) is melted. Proteins that bind to the double strand will tend to stabilize dsDNA, and melting will occur at higher forces. Proteins that bind to single stranded DNA (ssDNA) destabilize melting, provided that the rate of association is comparable to the pulling rate of the experiment. Many proteins, however, exhibit some affinity for both dsDNA and ssDNA. We describe experiments upon DNA + HMGB2 (box A), a nuclear protein that is believed to facilitate transcription. By characterizing changes in the structure of dsDNA with a polymer model of elasticity, we have determined the equilibrium association constant for HMGB2 to be K_ds = 0.15 ± 0.7 10⁹ M^-1 for dsDNA binding. Analysis of the melting transition reveals an equilibrium association constant for HMGB2 to ssDNA to be K_ss = 0.039 ± 0.019 10⁹ M^-1 for ssDNA binding.

Optical traps allow the single-molecule investigation of the chemo-mechanical properties of biomolecules¹. We have developed an ultra-stable optical trapping system capable of ångstr&diaero;m-level position resolution and used it to monitor transcriptional elongation by single molecules of E. coli RNAP polymerase (RNAP). This optical trapping system uses the anharmonic region of the trapping potential, where differential stiffness vanishes, to generate a force-clamp that operates without feedback-associated noise². We demonstrate methods of calibrating this anharmonic trapping region and strategies to eliminate common sources of noise associated with air currents. Records of transcriptional elongation obtained with this device showed discrete steps averaging ~3.7 Å, a distance equivalent to the mean rise per base found in B-DNA³. To determine the absolute position of the RNAP on the DNA template, we monitored transcription under conditions in which a single nucleotide species was held rate-limiting and then aligned the resulting transcriptional pauses with the occurrence of this rate-limiting species in the underlying template. The aligned pause patterns recorded from four molecules, each measured with a different ratelimiting nucleotide species, were used to determine the sequence of a short region of unknown DNA, demonstrating that the motion of a single processive nucleic acid enzyme may be used to extract sequence information directly from DNA⁴.

This paper reports an experimental study of the low laser intensity Kerr Effect produced by optical trapping of fluorescently labeled 100 nm diameter polystyrene particles in aqueous suspension. Optical trapping was made by a tightly focused and periodically blinking IR laser beam. A green laser beam, aligned co-linear with the IR laser, was used as the fluorescence excitation light. The fluorescence signals from particles trapped by the blinking IR laser were measured by a lock-in amplifier to improve the signal to noise ratio required to detect the very minute (sub-thermal fluctuation) changes in local particle density induced by optical trapping. The use of confocal detection ensured that the fluorescent signals measured were only from the diffraction-limited focal region of the two laser beams. By independently measuring the fluorescence intensity as a function of particle concentration and dn/dC (the change in refractive index due to change in concentration), we were able to determine the Kerr coefficients for laser trap powers in the range of 10.6 mW to 85 mW. Non-linear behavior in the refractive index vs. laser intensity relationship indicates that higher order Kerr coefficients are needed to describe the Kerr effect. Kerr coefficients obtained by using a circularly polarized IR laser were similar to those obtained by a linearly polarized laser, indicating that the induced electric dipole-dipole interactions did not contribute to the electric field-induced concentration changes giving rise to the Kerr effect.

Optical tweezers have been used extensively to measure the mechanical properties of individual biological molecules. Over the past 10-15 years optical trapping studies have revealed important information about the way in which motor proteins convert chemical energy to mechanical work. This paper focuses on studies of the acto-myosin motor system that is responsible for muscle contraction and a host of other cellular motilities. Myosin works by binding to filamentous actin, pulling and then releasing. Each cycle of interaction produces a few nanometres movement and a few piconewtons force. Individual interactions can be observed directly by holding an individual actin filament between two optically trapped microspheres and positioning it in the immediate vicinity of a single myosin motor. When the chemical fuel (adenosine triphosphate or ATP) is present the myosin undergoes repeated cycles of interaction with the actin filament producing square-wave like displacements and forces. Analysis of optical trapping data sets enables the size and timing of the molecular motions to be deduced.

Concentrated solutions of long polymer molecules exhibit reduced molecular diffusivity and striking non-Newtonian fluid properties that arise due to intermolecular entanglements. The most successful theories for describing these properties are based on the notion that on short time scales each polymer is confined to move within a tube-shaped region following its contour. Such a confining field is proposed to arise due to collective intermolecular interactions, yet this has remained a rather vague concept since the confining forces have never been directly measured. Here, we directly measure these forces by using optical tweezers to manipulate single entangled DNA molecules. We found that the forces opposing displacement of a molecule parallel to its local contour were negligible compared with those opposing transverse displacement. A time-dependent harmonic potential opposed transverse displacement. Work per unit length of order 1 kT was required to displace the molecule by a distance roughly equal to the theoretically predicted tube radius in the calculated thermal equilibration time. The required work also decreased gradually with the rate of displacement, in accord with predictions of a recent simulation study that found that the tube radius expands with time. Following the displacement, we measured the relaxation of force acting on the molecule and observed three distinct exponential decay times of ~0.4, 5, and 34 s, which are consistent with theoretically proposed molecular relaxation mechanisms. These measurements quantify the notion of a tube-like molecular confining field assumed in reptation theories.

We demonstrate a novel technique for creating, manipulating, and combining femtoliter to attoliter volume chemical containers. Possible uses include creating controlled chemical reactions involving small quantities of reagent, and studying the dynamics of single molecules. The containers, which we call hydrosomes, are surfactant stabilized aqueous droplets in a low index-of-refraction fluorocarbon medium. The index of refraction mismatch between the container and fluorocarbon is such that individual hydrosomes can be optically trapped by single focus laser beams, i.e. optical tweezers. Previous work on single molecules usually involved the tethering of the molecule to a surface, in order to interrogate the molecule for an extended period of time. The use of hydrosomes opens up the possibility for studying free molecules, away from any perturbing surface. We show that this is indeed true in the case of quantitative FRET with RNA. Furthermore, we demonstrate the controlled fusion of two hydrosomes for studying reactions, such as DNA binding kinetics, and single molecule dynamics under non-equilibrium conditions. We also show the applicability of our technique in analytical chemistry, such as for molecule identification and sorting.

We report on improvements and innovations in the use of hydrosomes to encapsulate and study single molecules. Hydrosomes are optically-trappable aqueous nanodroplets. The droplets are suspended in a fluorocarbon medium that is immiscible with water and has an index of refraction lower than water, so hydrosomes are stable and optically trapped by a focused laser beam (optical tweezers). Using optical tweezers, we hold the hydrosomes within a confocal observation volume and interrogate the encapsulated molecule by fluorescence excitation. This method allows for long observation times of a molecule without the need for surface immobilization or liposome encapsulation. We have developed a new way for creating hydrosomes on demand by inertially launching them into the fluorocarbon matrix using a piezo-activated micropipette. Time-resolved fluorescence anisotropy studies are carried out to characterize the effects of the hydrosome interface boundary on biological molecules and to determine whether molecules encapsulated within hydrosomes diffuse freely throughout the available volume. We measured the fluorescence anisotropy decay of 20mer DNA duplexes, and enhanced green fluorescent protein (GFP). We conclude that the molecules rotate freely inside the nanodroplets and do not stick or aggregate at the boundary.

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

Incorrect DNA repair is probably one cause of healthy ageing. Laser microbeams or optical tweezers are emerging as convenient tools in the study of repair mechanisms. Using such tools, DNA damage can be induced in a preselected volume element of a cell nucleus and at a preselected time point - an effect which is hardly to achieve with any other tool. On the other hand damage induction highly depends on a subtle combination of laser mircobeam parameters such as dose, pulse peak power and wavelength. In consequence DNA repair at the sites of damage may work differently. Furthermore, such sites are occasionally stationary, occasionally they migrate towards each other, indicating a considerable dynamics of DNA repair inside a cell nucleus. As an example for the application of optical tweezers, Erythrocyte Mediated Force Application (EMFA) is used to induce nitric oxide production in cells of the endothelium, i. e. the inner layer of (human) blood vessels. It is shown that upon stimulation by EMFA, endothelial cells initially activate the calcium homeostasis and develop calcium humps, concentration plateaus and oscillations.

We have developed a high-bandwidth technique for active 2-particle microrheology (AMR) with which we can probe linear and nonlinear responses of soft materials. Micron-sized colloidal probe particles are driven by an oscillating optical trap, and the resulting correlated motions of neighboring particles are detected by laser interferometry. Lock-in detection at the driving frequency and at its second harmonic makes it possible to measure the linear and the non-linear response of the embedding medium at the same time. We demonstrate the sensitivity of the method by detecting a second-harmonic response in water which is of purely geometric origin and which can be fully understood within linear hydrodynamics.

The red blood cell (RBC) viscoelastic membrane contains proteins and glycoproteins embedded in a fluid lipid bilayer that are responsible for cell agglutination. Manipulating RBCs rouleaux with a double optical tweezers, we observed that the cells slide easily one over the others but are strongly connected by their edges. An explanation for this behavior could be the fact that when the cells slide one over the others, proteins are dragged through the membrane. It confers to the movement a viscous characteristic that is dependent of the velocity between the RBCs and justifies why is so easy to slide them apart. Therefore, in a first step of this work, by measuring the force as a function of the relative velocity between two cells, we confirmed this assumption and used this viscous characteristic of the RBC rouleaux to determine the apparent membrane viscosity of the cell. As this behavior is related to the proteins interactions, we can use the apparent membrane viscosity to obtain a better understanding about cell agglutination. Methods related to cell agglutination induced by antigen-antibody interactions are the basis of most of tests used in transfusion centers. Then, in a second step of this work, we measured the apparent membrane viscosity using antibodies. We observed that this methodology is sensitive to different kinds of bindings between RBCs. Better comprehension of the forces and bindings between RBCs could improve the sensibility and specificity of the hemagglutination reactions and also guides the development of new potentiator substances.

Microrheology is the study of fluid flows and material deformations on a microscopic scale. The study of viscoelasticity of microscopic structures, such as cells, is one application of microrheometry. Another application is to study biological and medical samples where only a limited volume (microlitres) of fluid is available. This second application is the focus of our work and we present a suitable microrheometer based on optical tweezers. Optical tweezers are an optical trap created by a tightly focused laser beam. The gradient force at this focus acts to trap transparent micron sized particles, which can be manipulated within the surrounding environment. We use the polarisation of the incident field to transfer angular momentum to a trapped spherical birefringent particle. This causes the particle to rotate and measuring the polarisation of the forward scattered light allows the optical torque applied to the sphere to be calculated. From the torque, the viscosity of the surrounding liquid can be found. We present a technique that allows us to perform these measurements on microlitre volumes of fluid. By applying a time-dependent torque to the particle, the frequency response of the liquid can also be determined, which allows viscoelasticity to be measured. This is left as a future direction for this project.

Micro-optical components offer several possibilities for creating large matrices of optical traps, either when working on inverted microscopes, or by directly integrating miniaturized optical components at the level of a micro-fluidic chip. In this article we focus on two particular configurations, both allowing to generate large arrays of 3D optical traps. The first configuration takes advantage of an array of refractive microlenses to generate multiple optical tweezers within the focal plane of a high-NA microscope objective. The second configuration relies on an array of focusing high-NA micromirrors which are directly integrated at the level of a micro-fluidic chip. We also present measurements of the maximal optical trapping forces that can be reached with several types of cells commonly employed in biology and biotechnology, and demonstrate that these forces are essentially related to the bulk refractive index of the cells.

We have measured the optical force on isolated particles trapped in an optical lattice generated by the interference of two coherent laser beams by a method based on the equipartition theorem and by an independent method based on hydrodynamic-drag. The experimental results show that the optical force on a particle in this type of optical lattice depends strongly on the ratio of the particle diameter to the period of the lattice. By tuning this ratio, the force due to the optical lattice can be made to vanish. We also formed optical lattices involving two independent standing waves with different spatial periods formed by tightly focusing four laser beams which are pair wise coherent. By shifting the relative phases of the interfering beams we can advance the two waves in opposite directions. Depending on the spacing and the translation speed of the two interference patterns, appropriately sized particles can be translated in opposite directions; using this approach we succeeded in separating two different sizes of particles in the presence of a simulated fluid flow.

Authors propose an on-chip solution for continuous transport and separation of microspheres based on a lensless imaging technique, Talbot effect. High contrast intensity pattern of optical lattice is created at Talbot distance within the microfluidic chamber. The advantages of this proposed method include large-scale, power-efficient, compact, simple configuration etc. The binary two-dimensional chessboard grating was designed and fabricated. Experimentally regular array of microspheres were trapped with static optical lattice, while the translation of group of spheres were demonstrated with dynamic optical lattice. And continuous separation of microspheres with different sizes was realized. Finally, the experimental result of passive guiding of a group of spheres on a tilted asymmetric optical landscape was introduced.

Building on the ability to exert torques in optical tweezers, optically-driven rotating micromachines have reached the verge of practical application. Prototype devices have been made, and useful applications are being sought. We outline some general principles that can be applied to the design of optically-rotated devices, and describe a method for rigorous computational modelling that is well-suited to the optimization and engineering of such micromachines. Finally, we describe a method for rapid microfabrication of prototypes for testing, and some results of such tests.

With the aid of phase-only spatial light modulators (SLM), generalized phase contrast (GPC) has been applied with great success to the projection of binary light patterns through arbitrary-NA microscope objectives for real-time threedimensional manipulation of microscopic particles. Here, we review the analysis of the GPC method with emphasis on efficiently producing speckle-free two-dimensional grey-level light patterns. Numerical simulations are applied to construct 8-bit grey-level optical potential landscapes with high fidelity and optical throughput via the GPC method. Three types of patterns were constructed: geometric block patterns, multi-level optical trap arrays, and optical obstacle arrays. Non-periodic patterns were accurately projected with an average of 80% diffraction efficiency. Periodic patterns yielded even higher diffraction efficiencies, averaging 94%, by the utilization of large-aperture phase contrast filters.

In this paper, we make use of optically controlled phase sampling methods with the use of a dual beam optical trapping system. The dual beam trap enables us to remotely position a single birefringent particle and a second silica microsphere to form a Young's slits type experiments. The controlled rotation of the birefringent particle means that it acts as both a micro-diffuser and diffracting aperture at the same time. By observing the visibility of the far-field interference fringes, we were measure the relative mutual coherence of the optical field between any two selected point sources. This technique provides us with a unique localized sampling method.

Optical vortices became a hot topic since almost two decades ago, when it was recognized that Laguerre-Gaussian laser modes carry orbital angular momentum [Allen et al. Phys Rev A 45, 8185 (1992)] related with a screw phase dislocation, and different from the spin angular momentum associated to circular polarization. In 1995, this dynamical quantity was transferred to matter in an optical micromanipulation system for the first time [H. He, et al., Phys. Rev. Lett. 75, 826 (1995)], and since then, a number of studies on angular momentum of light have unveiled different interesting aspects on the subject. However, there are still open questions, which have arisen together with the generation of novel light beams, such as vector vortices, for instance. In contrast with scalar vortices, with usual polarization states (linear, circular, elliptical), the orientation and magnitude of the electric field of vector vortices (solutions of the vector wave equation) is a function of space and time. In this work, we present an experimental study of the local angular momentum density of a Bessel vector vortex of first order by means of an optical trap. For this purpose, we used different probe particles in order to sense the local contribution to the optical angular momentum in each region of the beam. But optical fields are not the only wave fields that may exhibit phase dislocations or singularities. There are close analogies between light and sound fields that can be exploited in order to get a better understanding of common phenomena and study new aspects in both branches of physics. Here we also present the first experimental demonstration and theoretical analysis of acoustical vortices in free field, with similar properties to those of the optical vortices, including the angular momentum that can be transferred to matter. The corresponding analogies and differences with the optical case turn out to be very enlightening for the understanding of the phenomenon of angular momentum in wave fields.

The intensity of spiral beams remains unchanged under propagation and focusing neglecting scaling and rotation. The spiral beam with predetermined intensity in the shape of any planar curve can be generated by use of amplitude and phase elements concurrently. We introduce the new method of singular laser fields formation, close to spiral type, by means of pure phase modulation. Our algorithm is based on the well-known Gerchberg-Saxton phase retrieval algorithm and spiral beams optics. It demonstrates fast convergence and some other advantages: phase distributions obtained are stable to spatial resolution changing (it is enough 128 x 128 pixels for some patterns), theoretical energy efficiency is about 85 % with acceptable intensity homogeneity. We demonstrate theoretical results on fields formation in the shape of closed-curves (triangular, square, "snowflake") and open-ended curve (Archimedes spiral) by means of elements on dichromate gelatin. Besides, the example of experiment on micromanipulation with the use of the square-shaped field is presented.

The longitudinal and transverse evolution of thermal clouds have been studied experimentally and theoretically in blue-detuned hollow tunnels. Tunnels based on axicon generation and holographic phase-mask generation have been investigated. A simple model is presented that (1) accounts for longitudinal acceleration and (2) shows that a cloud confined in a tunnel with a potential having a Bessel mode distribution will absorb fewer photons than it would confined in a comparable tunnel with a Laguerre-Gaussian mode distribution. The longitudinal and transverse profiles are fit to analytical distributions functions from which we extract transverse and longitudinal temperatures of the cloud. We find the two temperatures to be very different, with the transverse temperature being as much as five times colder. Finally, we studied the energy level structure within a Bessel potential theoretically and found that single-mode propagation is possible.

We present arguments in favor of the proposition that the momentum of light inside a transparent dielectric medium is the arithmetic average of the Minkowski and Abraham momenta. Using the Lorentz transformation of the fields (and of the coordinates) from a stationary to a moving reference frame, we show the consistent transformation of electromagnetic energy and momentum between the two frames. We also examine the momentum of static (i.e., time-independent) electromagnetic fields, and show that the close connection that exists between the Poynting vector and the momentum density extends all the way across the frequency spectrum to this zero-frequency limit. In the specific example presented in this paper, the static field inside a non-absorbing dielectric material turns out to have the Minkowski momentum.

We report our work of binding microscopic particles into three dimension structures through a novel layer-by-layer approach. Multi-beam interferometry was used to form a two dimensional periodic optical field. Such optical landscape was used to trap colloidal particles into a single layer of 2-d colloidal array. A second optical tweezers setup was combined to offset such single-layer 2-d colloidal arrays in the third dimension. A strong electric field was found at the vicinity of each single layer with carefully calculated experimental parameters. Our numerical results suggest that with practical parameters, the strong electric field resulted from the diffraction of trapped particles should trap colloidal particles into a new layer of identical 2-d arrays, thereby offering an alternative approach to optically bind microscopic particles into three dimension periodic structures. Preliminary experimental results of this method demonstrating trapping with secondary diffracted light are also presented.

We investigate near-resonant trapping of Rayleigh particles in optical tweezers. Although optical forces due to a near-resonant laser beam have been extensively studied for atoms, the situation for larger particles is that the laser wavelength is far from any absorption resonance. Theory predicts, however, that the trapping force exerted on a Rayleigh particle is enhanced, and may be three to fifty times larger for frequencies near resonance than for frequencies far off resonance. The ability to selectively trap only particles with a given absorption peak may have many practical applications. In order to investigate near-resonant trapping we are using nanoshells, particles with a dielectric core and metallic coating that can exhibit plasmon resonances. The resonances of the nanoshells can be tuned by adjusting the ratio of the radius of the dielectric core, r₁, to the overall radius, r₂, which includes the thickness of the metallic coating. Our nanoshells, fabricated at Rice University, consist of a silica core with a gold coating. Using back focal plane detection, we measure the trap stiffness of a single focus optical trap (optical tweezers), from a diode laser at 853 nm for nanoshells with several different r₁/r₂ ratios.

We demonstrate three-dimensional optical trapping and orientation of individual Au nanorods, Au/Ag core/shell nanorods, and Au bipyramids in solution, using the longitudinal surface-plasmon resonance to enhance optical forces. Laser light that is detuned slightly to the long-wavelength side of the resonance traps individual and multiple particles for up to 20 minutes; by contrast, light detuned to the short-wavelength side repels rods from the laser focus. Under stable-trapping conditions, the trapping time of individual particles depends exponentially on laser power, in agreement with a Kramers escape process. Trapped particles have their long axes aligned with the trapping-laser polarization, as evidenced by a suppression of rotational diffusion about the short axis. When multiple particles are trapped simultaneously, evidence of interparticle interactions is observed, including a nonlinearly increasing two-photon fluorescence intensity, increasing fluorescence fluctuations, and changing fluorescence profiles as the trapped particle number increases.

We discuss the application of optical trapping techniques to droplets, both in air (aerosols) and in fluid (emulsions). We show the holographic optical manipulation of aerosols and how this can be used to transfer orbital angular momentum to airborne particles. We demonstrate new types of traps for aerosols in the form of dual beam fibre traps and compare the trapping efficiency of IR and visible lasers. We discuss some of the interesting dynamics that can be observed when trapping airborne particles and how this appears to differ from conventional liquid based devices. We also examine how holographic optical trapping can be used to facilitate droplet manipulation in another liquid phase. We conclude with a discussion of the difficulties associated with trapping particles in air and possible solutions and well as look at some of the anticipated applications of such work, in particular in digital microfluidics.

The Brownian dynamics of an optically trapped water droplet is investigated across the transition from over to under-damped oscillations. The spectrum of position fluctuations evolves from a Lorentzian shape typical of overdamped systems (beads in liquid solvents), to a damped harmonic oscillator spectrum showing a resonance peak. In this later under-damped regime, we excite parametric resonance by periodically modulating the trapping power at twice the resonant frequency. We also derive from Langevin dynamics an explicit numerical recipe for the fast computation of the power spectra of a Brownian parametric oscillator. The obtained numerical predictions are in excellent agreement with the experimental data.

As a position sensing probe for Nano-CMM which measures three-dimensional shapes of microparts, we propose a novel probing technique using circular motion of an optically trapped microsphere. In this report, a fundamental principle is described for sensing a coordinate on a work surface using a circular motion probe. The circular motion of the trapped sphere near a work surface becomes an ellipse compressed perpendicularly to the surface due to the change of viscous drag of the sphere. The elliptical orbit of the trapped sphere depends on a distance from the surface and a normal vector direction of the surface. By processing the elliptical orbit, the circular motion probe can detect a position and a plane normal vector of the work surface simultaneously. In order to verify feasibility of this method, fundamental experiments are carried out. The circular motion probe is approached to a vertical silicon cleavage surface. The behavior of the trapped sphere near the surface agrees well with the theory. Based on the elliptical orbit of the trapped sphere near the surface, a position and a plane normal vector of the surface are estimated. It is verified that the circular motion probe can detect a position of a work surface with resolution of better than 50nm and detect a plane normal vector of the surface.

In this paper, we report a novel approach to functionalize the tip of a micro-cantilever by selectively positioning a functionalized polystyrene bead using optical tweezers as a lever arm. We present a design that consists of an Atomic Force Microscope (AFM) combined with an optical tweezers setup to study specific interactions between complementary protein molecules. A BSA protein coated polystyrene bead, held stationary in an optical trap, is chemically grafted to AFM cantliever tip functionalized by the complementary protein- anti BSA. This arrangement also provides a flexible means of reversibly and irreversibly fusing polystyrene beads thermally at desired specified locations on the micro-cantilever, by heating the silicon tip with the focused laser beam of the optical tweezers. We use optical tweezers as a micro-manipulation tool for grafting pre-specified number of beads to cantilever in a controlled fashion as against the other widely used methods where an aggregate of molecules are chemically attached. Further, we study the changes in cantilever's resonant frequency and find it in good agreement with the expected change due to the additional bead mass. This study opens up opportunities in the area of biosensors by providing a method to standardize the calibration of chemically modified cantilevers.

Laser induced cavitation bubbles in water results from either dielectric breakdown or the fast evaporation due to radiation absorption. The bubbles expand, reach a maximum radius and then supersonically collapse producing a by shock wave. So far, laser induced cavitation has been observed by using short pulsed (femto to nanosecond) lasers. In this report, we observe laser induced cavitation bubbles by using relatively low power (200 mW) cw light sources. A beam from a cw Nd:YAG laser (λ=1.064 μm) is tightly focused on saturated solution of copper nitrate salt. The large absorption coefficient at the illumination wavelength produces large thermal gradients and high peak temperatures leading to the formation of cavitation bubbles near the solution-glass interface. The collapse of the bubbles is so violent that they can be listened without any special device. Cavitation appears at quite regular time escales. The frequency of bubble formation depends on the laser intensity reaching frequencies as high as 4 KHz. We present detailed experimental results on the bubble formation using a phase contrast, light scattering and hydrophones.

We demonstrate how optical trapping and manipulation can be used to assemble microstructures. The microstructures we show being automatically recognized and manipulated are produced using the two-photon polymerization (2PP) technique with submicron resolution. In this work, we show identical shape-complementary puzzle pieces being manipulated in a fluidic environment forming space-filling tessellations. By implementation of image analysis to detect the puzzle pieces, we developed a system capable of assembling a puzzle with no user interaction required. This allows for automatic gathering of sparsely scattered objects by optical trapping when combined with a computer controlled motorized sample stage.

Optical Chromatography, used for particle separation, involves loosely focusing a laser into a fluid flowing opposite the direction of laser propagation. When microscopic particles in the flow path encounter this beam they are trapped axially along the beam and are pushed upstream from the laser focal point to rest at a point where the optical and fluid forces on the particle balance. Because optical and fluid forces are sensitive to differences in the physical and chemical properties of a particle, fine separations are possible. An optical chromatography beam which completely fills a fluid channel can operate as an optically tunable filter for the separation of polymeric/colloidal and biological samples. We will show how the technique can be used to separate injected samples containing large numbers of colloids. The power of optical chromatographic separations will be illustrated through the combination with other analytical techniques.

Pili are bacterial appendages that play many important roles in bacterial behaviors, physiology and interaction with hosts. Via pili, bacteria are able to adhere to, migrate onto, and colonize on host cells, mechanically. Different from the most studied type 1 and P type pili, which are rigid and thick with an average of 6~7 nm in diameter, type 3 pili are relatively tiny (3-5 nm in diameter) and flexible, and their biophysical properties remains unclear. By using optical tweezers, we found that the elongation processes of type 3 pili are divided into three phases: (1) elastic elongation, (2) uncoiling elongation, and (3) intrinsic elongation, separately. Besides, the uncoiling force of the recombinant pili displayed on the surface of E. coli [pmrkABCD_V1F] is measured 20 pN in average stronger than that of E. coli [pmrkABCD_V1]. This suggests that pilin MrkF is involved in determining the mechanical properties of the type 3 pili.

Optical tweezers are widely used for manipulating microscopic objects. Compared to other contact type microscopic manipulators such as micro-grippers that exhibit firm gripping, optical tweezers inherently possess loose gripping. For example, if a user tries to move target objects too fast such that the drag force of the viscous fluid exceeds the trapping force, target objects will escape from the effective trapping region. When this happens in a standard user interface environment with only video feedback, the user would sense this with a visual cue and slow down or slightly reverse the movement of the trap. In this study we enrich the user interface by adding a haptic cue that is a sense of forces and torques so that the user will sense the drag force and torque that is proportional to the gap distance and angle between the line trap and the nanowire. We present some preliminary results of putting haptic cue for manipulating nanowires.

Optically trapped microshperes can be manipulated by steering the trap beam, while the object position is measured with sub-nanometer resolution. A fast steering system is required to create feedback loop for measurements at a constant force or to increase position detection precision by trap stiffening. Using a real-time re-programmable digital signal processor, we combine steering and position detection to create a fast and versatile closed-loop feedback controlled instrument. We describe the construction and calibration of the instrument. We show that a proportional gain position-clamp algorithm can achieve about 10-fold increase in effective trap stiffness while higher gains lead to unwanted resonances.

By increasing the axial trap stiffness, we demonstrate an increase of at least 50% in the maximum lateral trapping force that can be applied using optical tweezers. It has previously been shown that, using a novel method of compensating for spherical aberrations, the axial trap stiffness at any particular chosen depth within a sample can be increased. However, to our knowledge, the present paper is the first time this method has been used in combination with the drag force method for the purpose of more accurately determining the maximum lateral trapping force applicable by optical tweezers. Previous studies have substantially shown that before the actual maximum lateral trapping force can be reached, the particle escapes in the axial direction. Using a conventional setup, our studies support this conclusion. However, by employing the above mentioned method for improving the axial trap stiffness, we observed that the displacement of the bead in the lateral direction is increased by approximately 10%. This allows progress towards a more accurate determination of the maximum lateral force that can be applied using optical tweezers and could also permit a mapping of the trapping potential further from the trap's central region. Theoretical predictions made, show that the point where the maximum lateral force could be applied is at 0.9 a, where a is the radius of the trapped particle. However, the experimentally measured limit 0.55 a has until now been far lower than that theoretically predicted 0.9 a. In this proceeding, we demonstrate that the experimental limit can be extended to 0.61 a because of the decreased axial displacement of the bead.

Oil immersion objectives have higher numerical aperture than water immersion objectives thus providing higher optical resolution. This is important for confocal microscopy as well as for the strength of an optical trap created by such an objective, because the efficiency of an optical trap is limited by its axial strength. However, light focused by oil immersion objectives suffer from spherical aberrations caused e.g. by a mismatch between the refractive index of the immersion and sample media. Such aberrations widen the intensity profile in the focal region thus restricting the axial resolution of the objective and decreasing the axial optical trapping strength. Objectives are typically designed such that the spherical aberrations are minimized for visible wavelengths and a few microns away from the surface. However, often optical traps are based on infrared lasers or are used further away from the surface thus introducing considerable spherical aberrations. We have shown that a tuning of the immersion refractive index can minimize the total spherical aberrations at any desired depth, thus maximizing the trapping efficiency and giving rise to optical trapping strengths twice as large as previously reported.¹ Changing the immersion media, however, is a discrete way of tuning the optimal trapping depth: An increment (decrement) of 0.01 in the refractive index of the immersion media gives rise to an increase (decrease) of ≃4 μm and ≃10 μm of the most efficient trapping depth for infinity-tube length and finite-tube length objectives, respectively. Here, we show that combining a change of immersion media with changing tube length provides a continuous way of changing the optimal trapping depth. Also, we show how trapping conditions change with polarization.

Laser cooling and trapping of atoms and molecules has been prevailing for two decades in numerous research fields and even in educational demonstrations. We propose here a novel application of laser cooling in the newly developed nanoparticle flame synthesis processes. With a robust laser cooling system, we could cool down specific species in a flame environment with high temperature and a variety of pressures. By combing with spontaneous Raman scattering and fluorescence, we could better diagnose and control the synthesis process in situ. The results were analyzed in the context of thermodynamics and reaction fluid to improve our knowledge on the mechanism of the gas-phase synthesis of nanoparticles.

We demonstrate the possibility to collect DNA molecules from solution on beads trapped by a focused laser beam close to the cell bottom. The combined action of laser-induced flow and laser pressure keeps the bead in place while the flow deposits the DNA. We show that the combined action of walls and volume absorption can control the direction and magnitude of the induced convention flow. We report trapping of viral bacterial DNA on micrometer size beads with laser power in the mW range.