We use optical tweezers to investigate processes happening inside ving cells. In a previous study, we trapped naturally occurring lipid granules inside living yeast cells, and used them to probe the viscoelastic properties of the cytoplasm. However, we prefer to use probes which can be specifically attached to various organelles within the living cells in order to optically quantify the forces acting on these organelles. Therefore, we have chosen to use nanometer sized gold beads as probes. These gold beads can be conjugated and attached chemically to the organelles of interest. Only Rayleigh metallic particles can be optically trapped and for these it is the case that the larger the beads, the larger the forces which can be exerted and thus measured using optical tweezers. The gold nanoparticles are injected into the cytoplasm using micropipettes. The very rigid cell wall of the S. pombe yeast cells poses a serious obstacle to this injection. In order to be able to punch a hole in the cell, first, the cells have to be turned into protoplasts, where only a lipid bilayer separates the cytoplasm from the surrounding media. We show how to perform micropipette delivery into the protoplasts and also how the protoplasts can be ablated using the trapping laserlight. Finally, we demonstrate that we can transform the protoplasts back to normal yeast cells.

Mechanical properties of living biological cells are important for cells to maintain their shapes, support mechanical stresses and move through tissue matrix. The use of optical tweezers to measure micromechanical properties of cells has recently made significant progresses. This paper presents a new approach, the oscillating optical tweezer cytorheometer (OOTC), which takes advantage of the coherent detection of harmonically modulated particle motions by a lock-in amplifier to increase sensitivity, temporal resolution and simplicity. We demonstrate that OOTC can measure the dynamic mechanical modulus in the frequency range of 0.1-6,000 Hz at a rate as fast as 1 data point per second with submicron spatial resolution. More importantly, OOTC is capable of distinguishing the intrinsic non-random temporal variations from random fluctuations due to Brownian motion; this capability, not achievable by conventional approaches, is particular useful because living systems are highly dynamic and often exhibit non-thermal, rhythmic behavior in a broad time scale from a fraction of a second to hours or days. Although OOTC is effective in measuring the intracellular micromechanical properties, unless we can visualize the cytoskeleton in situ, the mechanical property data would only be as informative as that of "Blind men and the Elephant". To solve this problem, we take two steps, the first, to use of fluorescent imaging to identify the granular structures trapped by optical tweezers, and second, to integrate OOTC with 3-D confocal microscopy so we can take simultaneous, in situ measurements of the micromechanics and intracellular structure in living cells. In this paper, we discuss examples of applying the oscillating tweezer-based cytorheometer for investigating cultured bovine endothelial cells, the identification of caveolae as some of the granular structures in the cell as well as our approach to integrate optical tweezers with a spinning disk confocal microscope.

Functionalized surfaces can affect (bio-) chemical reactions and control spatially the affinity for various binding partners. By either distributing specific binding points, by using biological reconstituted systems on the surface or by investigating whole cells, a surface phase relief is introduced. In the proximity of a trapped particle, these surface scatterers will both disturb the optical trap and the position tracking signal by changing the wave front of the trapping laser. We investigate the influence of an additional scatterer on trapping force, detection sensitivity and local viscous drag by scanning an optically trapped bead (probe) across a structured surface. Using a photonic force microscope, the probe's fluctuation traces are recorded interferometrically in three dimensions with nm precision and at scan rates of several hundred kilohertz with a quadrant photodiode. The phase disturbance located underneath the optical trap alters the interferometric probe position signals and can lead to incorrect interaction measurements. We propose and test a procedure to correct for the phase disturbance of the surface structure. In a roll over experiment, where one nano-sphere rolls over another, we prove the applicability of our phase correction approach. In addition we investigate the influence of small gold dots on the coverslip on trapping parameters which are relevant for specific interaction measurements in biotechnology.

In this work we developed a setup consisting of an Optical Tweezers equipped with linear and non-linear micro-spectroscopy system to add the capabilities of manipulation and analysing captured objects. Our setup includes a homemade confocal spectrometer using a monochromator equipped with a liquid nitrogen cooled CCD. The spectroscopic laser system included a cw and a femtosecond Ti:sapphire lasers that allowed us to perform Raman, hyper-Raman, hyper-Rayleigh and two photon Excited (TPE) luminescence in particles trapped with an Nd:YAG cw laser. We obtained Raman spectra of a single trapped polystyrene microsphere and a single trapped red blood cell to evaluate the performance of our system. We also observed hyper-Rayleigh and hyper-Raman peaks for SrTiO₃ with 60s integration time only. This was possible because the repetition rate of the femtosecond Ti:sapphire lasers, on the order of 80 MHz, are much higher than the few kHz typical picosecond laser repetition rate used before in hyper- Raman experiment, which required acquisition times of order of few hours. We used this system to perform scanning microscopy and to acquire TPE luminescence spectra of captured single stained microsphere and cells conjugated with quantum dots of CdS and CdTe and hyper-Rayleigh spectra of a noncaptured ZnSe microparticle. The results obtained show the potential presented by this system and fluorescent labels to perform spectroscopy in a living trapped microorganism in any neighbourhood and dynamically observe the chemical reactions changes in real time.

Living cells initiate a stress response in order to survive environmentally stressful conditions. We monitored changes in the Raman spectra of an optically trapped Saccharomyces cerevisiae yeast cell under normal and hyperosmotic stress conditions. When the yeast cells were challenged with a high concentration of glucose so as to exert hyperosmotic stress, it was shown that two chemical substances - glycerol and ethanol - could be monitored in real time in a single cell. The volume of the detection area of our confocal microspectrometer is approximately 1 fL. The average quantities of detected glycerol and ethanol are about 300 attomol and 700 attomol respectively. This amounts to the detection of approximately 10⁸ glycerol molecules and 4 X 10⁸ ethanol molecules after 36 min of hyper osmotic stress. Besides this, we also optically trapped a single yeast cell for up to three hours under normal conditions and monitored the changes in the Raman spectra during the lag phase of its growth and the G₁ phase of its cell cycle. During the lag phase the cell synthesises new proteins and the observed behavior of the peaks corresponding to these proteins as well as those of RNA served as a sensitive indicator of the adaptation of the cell to its changed environment. The changes observed in the Raman spectra of a trapped yeast cell in the late G₁ phase or the beginning of S phase corresponded to the growth of a bud.

Raman Tweezers Micro Spectroscopy has become an important and versatile technique in recent years. The technique is the amalgamation of optical tweezers and traditional Raman spectroscopy. The combination of these two well established techniques has brought key advantages in the studies of many different physical and biological systems from studying drug distribution in cells to measuring the size of aerosol particles. In this paper we present our Raman Tweezers system and discuss its advantages over conventional Raman systems, also discussed in this section is the parameters which effect collection of Raman scattered light using the ability of the optical tweezers to stack micro spheres. Finally we discuss how to extend further the functionality of the Raman tweezers technique by decoupling the trapping and excitation with the use of a fibre optical light force trap.

The goal of our study is to develop a setup that combines multi-trapping and manipulation with micro Raman spectroscopy of microns size particles. Multiple trapping, in 2D or 3D (two or three dimensional) configurations, is obtained in an inverted microscope scheme by shaping the trapping beam (1064 nm) with diffractive optical elements implemented on a spatial light modulator (SLM). Manipulation of multiple particles, directly trapped by the beam, can be achieved using the dynamic displaying of the SLM. Indirect trapping and manipulation of the sample can be obtained surrounding it with trapped micro beads that are manipulated by the optical tweezers. Laser light is not directly focused on the sample but is distributed on the beads and therefore the photo-induced damaging of biological samples is reduced. This technique offers also other advantages: the sample can be kept in a stable position during the spectroscopic investigation or can be moved in x-y-z to get spatial resolved information in a scanning mode measurement and the shape of a deformable sample can be changed in a controlled manner during the measurement. Sample's excitation and Raman signal collection are accomplished with a separate laser beam (514.5 nm) in a non-inverted microscope coupled with the spectrometer. Some experimental results showing multi trapping and indirect manipulation of human red blood cells are presented and discussed.

Up to now optical spectroscopies have analyzed the scattered light or the heat generated by absorption as a function of the wavelength to get information about the samples. Among the light matter interaction phenomena one that has almost never been used for spectroscopy is the direct photon momenta transfer. Probably because the forces involved are very small, varying from hundreds of femto to tens of pico Newtons. However, the nowadays very popular Optical Tweezers can easily accomplish the task to measure the photon momenta transfer and may be the basis for the Optical Force Spectroscopy. We demonstrate its potential as such a tool by observing more than eight Mie resonance peaks of a single polystyrene microsphere, and showed the capability to selective couple the light to either the TE, TM or both microsphere modes depending of the beam size, the light polarization and the beam positioning. The Mie resonances can change the optical force values by 30-50%. Our results also clearly show how the beam polarization breaks the usually assumed azimuthal symmetry by Optical Tweezers theories. We also obtained the spectrum from the two photon excited luminescence using the Optical Tweezers to hold a single bead suspended and a femtosecond Ti:sapphire laser for the non-linear excitation. This spectrum shows the pair of peaks due to both TE and TM spherical cavity modes. We have been able to observe more than 14 Mie resonance peaks in the TPE luminescence. Our results are in good agreement with optical force calculations using Maxwell stress tensor and partial wave decomposition of the incident beam approximated to a 3^th order gaussian beam.

As a powerful and noninvasive tool, laser trapping has been widely applied for the confinement and physiological study of biological cells and organelles. Researchers have used the single spot laser trap to hold individual sperm and quantitatively evaluated the motile force generated by a sperm. Early studies revealed the relationship between sperm motility and swimming behavior and helped the investigations in medical aspects of sperm activity. As sperm chemotaxis draws more and more interest in fertilization research, the studies on sperm-egg communication may help to explain male or female infertility and provide exciting new approaches to contraception. However, single spot laser trapping can only be used to investigate an individual target, which has limits in efficiency and throughput. To study the chemotactic response of sperm to eggs and to characterize sperm motility, an annular laser trap with a diameter of several hundred microns is designed, simulated with ray tracing tool, and implemented. An axicon transforms the wavefront such that the laser beam is incident on the microscope objective from all directions while filling the back aperture completely for high efficiency trapping. A trapping experiment with microspheres is carried out to evaluate the system performance. The power requirement for annular sperm trapping is determined experimentally and compared with theoretical calculations. With a chemo-attractant located in the center and sperm approaching from all directions, the annular laser trapping could serve as a speed bump for sperm so that motility characterization and fertility sorting can be performed efficiently.

Swimming activity of flagella is a main factor of the motility of bacteria. Flagella expressed on the surface of bacterial species serve as a primary means of motility including swimming. We propose to use optical tweezers to analyze the swimming activity of bacteria. The sample bacteria in the work is Pseudomonas aeruginosa, and it is a gram-negative bacterium and often causes leading to burn wound infections, urinary-tract infections, and pneumonia. The single polar flagellum of P. aeruginosa has been demonstrated to be important virulence and colonization factor of this opportunistic pathogen. We demonstrate a gene to regulate the bacterial swimming activity in P. aeruginosa PAO1 by biological method. However, the change of flagellar morphology was not observed by electron microscopy analysis, suggesting that the gene regulates the flagellar rotation that could not be detected by biological method. PFM exhibits a spatial resolution of a few nanometers to detect the relative position of the probe at an acquisition rate over 1 MHz. By binding a probe such as a bead or a quantum dot on the flagella, we expect the rotation of the probe due to the flagella could be detected. It is expected that the study of the swimming activity of P. aeruginosa provide potent method for the pathogenic role of the flagella in P. aeruginosa.

Optical tweezers are proving themselves useful and powerful tools in many areas of biological research, including fungal cell biology. Here we investigate their potential as a novel method for measuring the growth forces of fungal hyphae. We have calibrated the trapping forces produced by our tweezers system, and using beads have investigated whether growing hyphae can overcome this force and push beads out of the trap. Although 4 μm beads were pushed out of the trap by fungal hyphae, using 1 μm beads produced some more intriguing results. The problems encountered with this method of measurement and possible solutions are discussed.

Cells that are exposed to varying amounts of ultrasonic energy in the presence of ultrasound contrast agent (UCA) may undergo either permanent cell membrane damage (lethal sonoporation), or a transient enhancement of membrane permeability (reversible or non lethal sonoporation). The merits of each mode are clear; lethal sonoporation constitutes a significant tumour therapy weapon, whilst its less intrusive counterpart, reversible sonoporation, represents an effective non-invasive targeted drug delivery technique. Our working hypothesis for understanding this problem was that the root cause and effect in sonoporation involves the interaction of individual cells with single microbubbles, and to that end we devised an experiment that facilitates video rate observation of this specific scenario under well defined optical control. Specifically, we have constructed an innovative hybridization apparatus involving holographic optical trapping of single and multiple UCA microbubbles, together with the facility to irradiate with MHz pulsed ultrasound energy in the presence cancerous cells. This approach allows the isolation of a target microbubble from a resident population and the relocation to a [controllable] predetermined position relative to a cell within a monolayer. Frame extraction from standard framing rate video microscopy demonstrates the individuality of single microbubble-cell interactions. We describe a fluorescence microscopy protocol that will allow future study of the potential to deliver molecular species to cells, the dependence of the delivery on the initial microbubble-cell distance and to determine the targeted cell survival.

Suspensions of motile E. coli bacteria serve as a model system to experimentally study the hydrodynamics of active particle suspensions. Colloidal probe particles are localised within a suspension of motile bacteria by use of optical tweezers and their uctuations are monitored. The activity of the bacteria effects the fluctuations of the probe particles and their correlation, revealing information about the hydrodynamics of the suspension. We highlight experimental problems that make the interpretation of 'single probe' experiments (as reported before in literature) diffcult and present some preliminary results for 'dual probe' cross-correlation experiments.

Recent experimental and theoretical progress in the studies of Bose-Einstein condensation (BEC) has precipitated an intense effort to understand and control interactions of nonlinear matter-waves. Key ingredients in manipulations of matter-waves in BECs are a) external localized impurities (generated by focused laser beams) and b) periodic potentials (generated by interference patterns from multiple laser beams illuminating the condensate). In this work we demonstrate the ability of time-dependent external optical potentials to drag, capture and pin a wide range of localized BEC states, such as dark and bright solitons. The stability and existence of pinned states is analyzed using perturbation techniques, which predict results that are well corroborated by direct numerical simulations. The control of these macroscopic quantum states has important applications in the realm of quantum storage and processing of information, with potential implications for the design of quantum computers.

Optical binding may arise due to interplay between light scattering and refraction creating equilibrium positions for particles in a self-consistent manner. Binding is observed for the first time in biological cells within a dual beam fiber trap.

We report the first direct experimental mapping of three-dimensional optical force field on a silica micro-particle trapped in a counter-propagating dual-beam trap. We tracked the three-dimensional Brownian motion of the trapped particle (by optical position sensing) and analyzed the particle position distribution to obtain the force constant of the optical force field on the particle along each direction. The trapping beams scattered by the trapped particle along two directions (mutually orthogonal to each other and also to the trapping beams) were projected on a pair of quadrant photo-detectors (QPDs) to facilitate high-speed (20 KHz) three-dimensional position tracking. Position tracking over two mutually orthogonal planes intrinsically provides one set of redundant data for a self-consistency check. At optical wavelength λ = 532nm, the force constants of the three-dimensional optical force field on a silica micro-particle (diameter = 2.58μm) were determined to be k_x = 1.61×10^-1 pN/μm, k_y = 1.49×10^-1 pN/μm, and k_z = 4.43×10^-2 pN/μm when the total trapping power was about 21mW and the distance between the two fiber end-faces was 125μm. The set of force constants (k_x, k_y, and k_z) completely defines the optical force field E(x, y, z) = [k_xx² + k_yy² + k_zz²]/2 (in the parabolic potential approximation) on the trapped particle.

A detailed distribution of the force of electromagnetic radiation in and around dielectric media can be obtained by a direct application of the Lorentz law of force in conjunction with Maxwell's equations. We develop a theory of the force exerted by a focused light beam on the free surface as well as within the volume of a transparent dielectric medium. Although the medium can be either solid or liquid, here we emphasize the application of the formulas to liquids since, in principle at least, surface deformations and liquid motions are measurable. Our theory predicts that, upon entering the liquid from the free space, the beam of light exerts an outward vertical force on the entrance surface that tends to produce a localized bulge. This surface force, however, is much weaker than that predicted by prevailing theories and, in contrast to current beliefs, is found to depend on the polarization state of the incident beam. Within the volume of the liquid we predict that the forces of radiation tend to create four counter-rotating vortices at the four corners of the focused spot; the sense of rotation within these vortices depends on the direction of the incident polarization. These striking departures from conventional wisdom with regard to the force of radiation arise from a revision in the form of the Lorentz law as applied to the bound charges/currents within a dielectric medium.

The bending of fluid interfaces by the optical radiation pressure is now recognized as an appealing contactless tool to probe microscopic surface properties of soft materials. However, as the radiation pressure is intrinsically weak (typically of the order of a few Pascal), investigations are often limited to the regime of weak deformations. Non-linear behaviors can nevertheless be investigated using very soft fluid interfaces. Either a large stable tether is formed, or else a break-up of the interface occurs above a well-defined beam power threshold, depending on the direction of the beam propagation. This asymmetry originates from the occurrence of total reflection condition of light at deformed interface. Interface instability results in the formation of a stationary beam-centered liquid micro-jet that emits droplets. Radiation-induced jetting can also lead to giant tunable liquid columns with aspect ratio up to 100, i.e. well beyond the fundamental Rayleigh-Plateau limitation. Consequently, the applications range of the opto-hydrodynamic interface instability is wide, going from adaptative micro-optics (lensing and light guiding by the induced columns) to micro-fluidics and microspraying, as fluid transfer is optically monitored and directed in three dimensions.

In this paper, we report on near field optical tweezers using focused evanescent illumination. The intensity pattern of the focused evanescent field has been characterized using a SNOM. The near field trapping efficiency has been experimentally and theoretically investigated using both TEM₀₀ and TEM₀₁ beams. The trapping efficiency was found to be polarization dependent and also changed for the two modes of illumination used. By incorporating the near field and far field optical tweezers one could construct an optical micromanipulation setup with great flexibility and accuracy, which would find applications in optical nanometry. By coupling the techniques like morphology dependent resonance (MDR), one could employ near field tweezers for near field sensing and for characterization of microfluidic channels.

Recently a non-contact organization of submicron colloidal particles on the surface attracted a great attention in connection with development of imaging techniques using total internal reflection. We focus here on the theoretical description of the forces acting on a submicron particle placed in an interference field created by two counter-propagating evanescent waves. Numerical results elucidate how these forces or trap depth depend on the particle size and angle of incidence of both beams. Experimental results proved these conclusions and several polystyrene particles of diameter 520 nm were confined in evanescent standing wave.

The Anti-Brownian Electrophoretic trap (ABEL trap) allows a user to trap and manipulate individual fluorescent molecules in solution. The heart of the ABEL trap is a microfluidic cell. In previous incarnations of the ABEL trap, the microfluidic cell was formed from a polydimethylsiloxane (PDMS) stamp and a glass coverslip. Here we present an improved microfluidic cell, made entirely out of glass. This new design significantly decreases the rate of photobleaching, which previously limited the time that a single molecule could be trapped. Chemical modifications to the surface of the cell prevent adsorption and allow one to control the balance between electroosmotic and electrophoretic forces. The depth of the trapping region in the cell can be adjusted to allow trapping of different-sized objects.

When near-infrared laser was focused in colloidal silver, not only optical trapping of silver nanoparticles, but also hyper-Rayleigh scattering were observed at the laser focus. By addition of NaCl and rhodamine 6G in the colloidal silver, hyper-Rayleigh scattering was enhanced and hyper-Raman scattering was also observed. These nonlinear optical responses showed temporal fluctuation in spite of the continuous laser irradiation. Experimental results suggested that aggregates which have a high activity for nonlinear optical responses were trapped and/or produced by a focusing near-infrared laser. Hyper-Raman signal, whose scattering cross section is much lower than normal Raman scattering, could be obtained easily by focusing a cw-YAG laser in colloidal silver including analytes. It was demonstrated that optical trapping of colloidal silver is a powerful technique to obtain the nonlinear optical responses.

A strong electric field enhancement was found at the center of an evanescent wave centripetally-propagated along the surface of a multilayer dielectric waveguide coating on a conical prism. Behaviors similar to the cylindrical vector polarization was observed and enhanced for three orders of magnitude in the same geometry by reconfiguring the incident angles. This enhanced electric field can be utilized to trap dielectric particles with a refractive index either higher or lower than the surroundings. The enhanced optical-trap, also versatile at various incident angles, was analyzed numerically with Rayleigh scattering approximation. Simulation results for optical trapping application are presented, showing a strong gradient force on the trapped particle yet keeping radiation force negligible.

Optical tweezers technique has aggrandized the light-matter interaction, whereby researchers have employed light forces from colloidal sciences to biomedical applications. There have been constant quests to integrate novel beams in tweezers system for myriads of novel applications. In this paper, we report optical tweezers system based on self-imaged optical bottle beams, which has some distinct advantages over other conventional beams like Gaussian, Laguerre-Gaussian and Bessel beams in few applications. A self-imaged bottle beam is obtained by superimposing two Bessel beams with varying radial wavevectors. A self-imaged bottle beam possesses three-dimensional intensity-null points along the propagation axis and by varying the radial wavevectors of the Bessel beams number of bottles; location of bottles and the dimension of the bottles can be pre-determined. Moreover, by dynamically varying the parameters of radial wavevector of interfering Bessel beams, the dynamic potential-well obtained are capable of transporting both high and low indices microparticle in conveyor-belt arrangement along the longitudinal axis. Bottle beams belongs to the family of propagation-invariant beams and hence inherent the propagation-invariance and self-reconstruction properties of Bessel beam together with three-dimensional trapping potential contrary to Bessel beam whereby the particles trapped in the central core are loosing defined in the longitudinal axis. Such bottle beams also holds promise applications in blue-detuned optical taps for the optical confinement of laser-cooled atoms in three-dimensional dark region.

We demonstrate the dynamic properties of the optically vibrated microsphere for establishing a position detection probe for the nano-CMM. The principle of the position detection probe is based on the single-beam gradient-force optical trap of a micrometer size probe sphere and the vibration probing technique. An optically trapped microsphere in air is forced to vibrate in the transverse direction using the oscillating Nd: YAG laser beam. By studying the frequency response of the optically vibrated microsphere using PSD, the dynamic properties such as spring constants in the different directions are evaluated. Both numerical and experimental analyses are performed to investigate the relation between a viscous drag coefficient and a distance from a surface of a specimen to be measured. Based on the obtained information, a novel principle of position sensing using the phase delay response due to drastically increasing viscous drag coefficients near the surface is proposed. Fundamental measurement of the phase delay is demonstrated by probing the edge of an as-cleaved silicon wafer in the lateral direction. The measurement results suggest that the proposed method is valid for sensing a position with the resolution of nanometer order.

In this paper we present the standing wave created from two counter-propagating non-diffracting (Bessel) beams as a device for confinement and precise delivery of sub-micron sized particles. The particle position in direction of beam propagation is controled by changing the phase shift between these two beams. We succeeded in delivery of polystyrene particles of diameter 410 nm over a distance of 300 μm. At the same time we experimentaly confirmed the theoretical prediction how the optical forces acting on particles in this kind of field depend on the size of the objects .

Shaping optical fields is the key issue in the control of optical forces that pilot the manipulation of mesoscopic polarizable dielectric particles. The latter can be positioned according to endless configurations. The scope of this paper is to review and discuss several unusual designs which produce what we think are among some of the most interesting arrangements. The simplest schemes result from interference between two or several coherent light beams, leading to periodic as well as pseudo-periodic arrays of optical traps. Complex assemblages of traps can be created with holographic-type set-ups; this case is widely used by the trapping community. Clusters of traps can also be configured through interferometric-type set-ups or by generating external standing waves by diffractive elements. The particularly remarkable possibilities of the Talbot effect to generate three-dimensional optical lattices and several schemes of self-organization represent further very interesting means for trapping. They will also be described and discussed. in this paper. The mechanisms involved in those trapping schemes do not require the use of high numerical aperture optics; by avoiding the need for bulky microscope objectives, they allow for more physical space around the trapping area to perform experiments. Moreover, very large regular arrays of traps can be manufactured, opening numerous possibilities for new applications.

The generalized phase contrast (GPC) method is used to transform a collimated Gaussian beam into a manifold of counterpropagating-beam traps capable of real-time interactive manipulation of a plurality of particles in three dimensions. Contrary to high-NA based optical tweezers, GPC can operate with a centimeter long working distance, avoiding the use of objective lens immersion liquid and offers a wider manipulation region and a larger field of view than optical tweezers. As a consequence of the large working distance, simultaneous monitoring of the trapped particles in orthogonal observation planes can be easily integrated. Real-time 3D reconfigurable light patterns with sub-micron accuracy are obtained from a direct map of phase patterns addressed on phase-only spatial light modulator followed by a programmable parallel polarisation modulating device. A graphical user-interface enables interactive and arbitrary control over the dynamics and geometry of the real-time synthesized light patterns. Experimental demonstrations have shown that GPC-driven micro-manipulation can be used for fully user-guided assembly of particles in a volume, control of particle stacking along the optical axis, manipulation of multiple low-index particles, real-time sorting of inhomogeneous mixtures of micro-particles and the organization of living cells into 3D reconfigurable colloidal structures.

Studying the thermal fluctuations of DNA molecules reveals not only a wealth of interesting equilibrium and nonequilibrium statistical mechanics, but is also of importance for understanding the dynamics of DNA in vivo. An instance of the latter is in the context of regulatory functions that require collaborative interactions of distant operator sites on the DNA molecule. These thermal fluctuations are extremely sensitive to mechanical constraints, such as supercoiling or mechanical tension in the DNA. The natural force scale fc on which these fluctuations are sensitive to tension is related to the persistence length l_p by f_c = k_BT/l_p = 80 fN, which is generally considered small for a crowded cellular environment. We are studying the dynamics of single DNA molecules under tension under equilibrium and non-equilibrium conditions using a modified scanning-line laser trap. This technique allows us to apply a constant force between 20 fN and 3 pN to a λ-DNA molecule while we study its behavior under two different conditions: 1) nonequilibrium studies in which we observe the relaxation trajectory of a highly extended DNA molecule as it returns to its equilibrium conformation against an applied optical force, and 2) equilibrium studies which measure fluctuations in the extension of the molecule about its mean with sub-millisecond time resolution as a function of its extension. In the nonequilibrium studies we find a marked deviation from predictions derived from the wormlike-chain (WLC) model for extended DNA molecules. In the equilibrium studies we compute the time-correlation functions of the fluctuations to determine their time constants, and model them with a simple bead-and-spring model. We observe a decrease of the fundamental time constant with increasing extension of the molecule. This suggests that the change in spring constant dominates changes in the intra-chain hydrodynamic coupling between segments as the Gaussian coil unravels into an extended conformation.

We measure the entropic elasticity of a single λ-DNA molecule and of a single DNA-Psoralen complex by doing stretching experiments with an optical tweezers. Psoralen is a photosensitive drug used in the treatment of many skin diseases, by impeding DNA replication. Psolaren intercalates the DNA and can form crosslinks with pyrimidine basis in opposite strands of DNA, when illuminated with UVA light. As crosslinks form the persistence length of the complex increases, indicating an increase in rigidity of the complex. We study the kinetics of DNA-Psoralen crosslink formation via changes in entropic elasticity of the complex.

Mechanical manipulation of single DNA molecules can provide novel information about protein-DNA interactions. Here we review two examples studied by our group. First, we have studied the forced unraveling of nucleosomes assembled on heterogeneous DNA using core histones, the histone chaperone NAP-1, and ATP-dependent chromatin assembly and remodeling factor (ACF). We measure abrupt events releasing ~55 to 95 base pairs of DNA, which are attributable to non-equilibrium unraveling of individual nucleosomes. Wide variations observed in the unraveling force and sudden DNA re-wrapping events may have an important regulatory influence on DNA directed biochemical processes. Second, we have studied the mechanics and dynamics of single DNA looping and cleavage by "two-site" restriction enzymes. Cleavage is measured as a function of DNA tension, incubation time, and enzyme concentration, distinguishing enzymes that require DNA looping from ones that do not. Forced disruption of fixed DNA loops formed in the absence of Mg²⁺ is observed, allowing the distribution of number of loops, loop length, and disruption force to be measured as a function of time, DNA tension, and ionic conditions.

Here we describe and characterize a method for manipulating desired DNA sequences from any organism with optical tweezers. Molecules are produced from either genomic or cloned DNA by PCR using labeled primers and are tethered between two optically trapped microspheres. We demonstrate that human, insect, plant, bacterial, and viral sequences ranging from ~10 to 40 kbp can be manipulated. Force-extension measurements show that these constructs exhibit uniform elastic properties in accord with the expected contour lengths for the targeted sequences. Detailed protocols for preparing and manipulating these molecules are presented, and tethering efficiency is characterized as a function of DNA concentration, ionic strength, and pH. Attachment strength is characterized by measuring the unbinding time distribution as a function of applied force.

We report here on the use of optical tweezers in the growth and manipulation of protein and inorganic crystals. Sodium chloride and hen egg-white lysozyme crystals were grown in a batch process, and then seeds from the solution were introduced into the optical tweezers. The regular and controllable shape and the known optical birefringence in these structures allowed a detailed study of the orientation effects in the beam due to both polarization and gradient forces. Additionally, we determined that the laser tweezers could be used to suspend a crystal for three-dimensional growth under varying conditions. Studies included increasing the protein concentration, thermal cycling, and a diffusion-induced increase in precipitant concentration. Preliminary studies on the use of the tweezers to create a localized seed for growth from polyethylene oxide solutions are also reported.

Aerosol droplets are trapped and manipulated with a single-beam gradient-force optical trap for timescales of hours. By coupling the optical trap with cavity enhanced Raman scattering, the size of the trapped droplet can be determined with nanometre accuracy and high time resolution. This allows the evolution in droplet size and composition to be monitored during the growth or evaporation of a single trapped droplet, providing a method for characterising the factors that govern aerosol droplet size. The simultaneous trapping of two or more aerosol droplets in parallel optical traps can permit studies of aerosol coagulation.

Colloidal systems form the basis of many complex areas of academic and industrial research efforts. As well as contributing to the understanding of many industrially-produced substances such as paints and glues, they have also proved an excellent model thermodynamic system. Changing the properties of an ensemble of colloidal particles, chemically or otherwise, and observing the evolution of the many-body system gives insight into thermodynamic phenomena such as condensation and crystallisation. Some colloidal systems with a particle density that would place them around the dense end of the liquid-crystal coexistence region show a transition into an amorphous glassy state. Long range particle movement is prevented, while local diffusion is still allowed. Such systems are ideal candidates for exploration with optical force measurements, which allow the relatively non-intrusive manipulation of particles deep within colloidal suspensions. Careful use of refractive index matching has allowed an invisible colloidal suspension to be examined with probe particles of a similar size, but higher refractive index. The environment of particles in colloidal cages has been measured by studying the forces acting on a localised particle, as well as the forces needed to break one or more cages.

The analogous nature of dislocations in crystals and light leads to some interesting links between the properties of optical and crystal dislocations. A dislocation in a crystal can be described by a Burgers vector b, whilst a dislocation in a laser mode such as the vortex in a Laguerre Gaussian beam, can be described by a topological charge l². By illuminating both optically trapped and self-assembled two-dimensional colloidal crystals with a Gaussian laser beam, we show a direct link between crystal and light dislocations, where the first order diffraction pattern from a crystal with Burgers' vector b = na contains vortex laser modes of topological charge l=±mn, (where n is an integer, a is the lattice constant of the crystal and m is an integer corresponding to the diffraction order).

Laser-photophretic behavior of the micrometer-sized single droplets of photo-absorbing microemulsion was observed in water by irradiating a cw Ar⁺ ion laser. We have made an unexpected observation that the droplets repeated the expansion and the sudden contraction motion during the laser photophoretic migration. The period of the expansion-contraction cycle was decreased as the irradiated laser power was increased and was independent of the initial size of the droplet. The origin of the cyclical motion is the temperature rise of the droplet due to the photo-thermal conversion. The three-step mechanism for the periodic motion of the droplet was proposed. The first step is the phase separation of the bicontinuous microemulsion droplet into the normal w/o microemulsion outer phase and the aqueous inner phase in the droplet, which was caused by the laser-induced temperature gradient inside the droplet. In the second step, an expansion of the inner aqueous phase was induced by the percolation of the external water by thermo-osmosis, which was caused by the laser-induced temperature gradient between inside and outside of the microemulsion liquid membrane of the droplet. In the third step, the liquid membrane became thinner to a critical thickness which induced its break and the droplet shrank into the original shape. The proposed mechanism for the expansion process was confirmed by the numerical simulation of the volume change of the droplet during the laser irradiation, taking account of the heat evolution in the droplet, the heat transfer from the droplet, and the percolation of the external water by thermo-osmosis and by the high-speed CCD imaging of the contraction motion of the droplet.

Near-field optical manipulation has emerged as an alternative technique for trapping and manipulation of small particles (micro- and nano-objects) due to the localization of the field to typically less than 100nm from an interface. It has been demonstrated that near-field photonic forces can move microparticles located near an interface. The surface field couples to microparticles in close proximity, where the near-field wave can be converted to a propagating wave, via photon tunnelling across the gap from prism to microparticles. We have extended this method to observe both guiding and trapping of microparticles in unison over an extended area of 1mm² in a system we call Lensless Optical Trapping. Our method makes use of a patterned evanescent field that produces well defined individual potential wells where objects immersed in water can be laterally trapped and longitudinally guided along the propagation direction of the evanescent waves. Stable trapping in an array was achieved by using two counterpropagating beams, balancing the forces involved in the optical trapping. Our method permits full control of speed, direction and position of thousands of microparticles simultaneously. This will be useful for future lab-in-a chip cell sorting. Evanescent electric field can be increased until two orders of magnitude by using a metallic surface. We also present the first demonstration of an enhancement of the accumulation of nanoparticles by optically exciting surface plasmons. Convection and thermophoresis effects are also involved in this accumulation and the role of the thermal and optical forces involved will be discussed in detail.

Two counter-propagating laser beams incident at the silica-water interface above the critical angle set up interference fringes (with a spacing of 0.4-μm) in an evanescent wave. This paper describes the optical binding of sub-micron polystyrene spheres in the evanescent field. At low particle densities, the particles form lines perpendicular to the fringes; the particle positions may be commensurate or incommensurate with the fringes, depending on the particle size. At higher particle densities, well-ordered 2-D arrays form with a centred rectangular lattice in which the lattice constant in the plane of incidence is twice the fringe spacing. Rotating the plane of polarisation of one of the two laser beams eliminates the fringes, but ordered arrays still form. These arrays are either rectangular or pseudo-hexagonal depending on the particle size. A range of dynamical behaviour analogous to that of atomic crystals was observed.

We report quantitative measurements of the radiation forces exerted on a micrometer dielectric sphere by a Surface Plasmon Polariton (SPP) excited at a gold/water interface. We separate the contributions of the two constituents of the plasmon wave - the electromagnetic field and the charge-density oscillations - to the total radiation force. Measurements performed with a Photonic Force Microscope (PFM) show an enhanced attraction to the surface compared to a conventional evanescent wave at the dielectric interface (10² enhancement factor).

We have shown earlier that photopolymerization offers a relatively simple method to produce microscopic particles of arbitrary shape that are practical to expand the possibilities of optical manipulation. Propeller shaped micrometer sized rotors are rotated in optical tweezers, while flat objects are oriented in traps formed by linearly polarized light. Such elements and the possibilities opened up by their use would find numerous applications in lab-on-a-chip devices. We have extended these methods by developing new elements for applications. We have built microscopic wheels ad gears that have a central flat component so that its rotational position can be controlled by linearly polarized light. These rotors are rotated by rotating the polarisation of the light. Such gears can be used as actuators of more complex micromechanical devices (pumps, valves,) also built by photopolymerization in a single process. Important component of such devices are gears rotating on fixed axes, readily built by the method. An alternative way of optically actuating rotating devices by light is the illumination of cogwheel shaped rotors from a tangential direction. The advantage of this latter approach is that the whole system (i.e. the rotors on axes and optical waveguides that carry the actuating as well as possibly the sensing light) can be built as an integrated system in a single process. Such devices would not need optical tweezers and thus bulky microscopes for actuation, significantly reducing the complexity of eventual lab-on-a-chip devices. Operational examples will be demonstrated and the properties of the different approaches will be compared.

In the emergent field of microfluidics there is the desire to process ever decreasing amounts of analyte. This will challenge our ability to pump, move and mix small volumes of fluid and will require the controlled actuation of various microcomponents. A valuable tool for micromanipulation is optical tweezing. Here the forces due to the reflection and refraction of light at a dielectric interface can be used to pick up, move and rotate microscopic particles. A powerful method of rotating microparticles at high rates in optical tweezers is the use of birefringence. A birefringent particle can be rotated in a standard optical trap simply by manipulating the input light's state of polarisation, however this requires specialist materials that are not easily micromachined. Here we present the modelling, fabrication and rotation of form birefringent microgears. The birefringence comes from the shape of the microgear (a 1D photonic crystal etched into the center of the microgear) rather than the materials intrinsic properties allowing us to use materials convenient for microfabrication. We have demonstrated the orientation of microgears with the direction of polarisation of linearly polarised light and rotation by rotating the direction of polarisation of linearly polarised light and by illumination with circularly polarised light. We have modelled and experimentally measured the magnitude of the birefringence with good agreement. We have also demonstrated that one microgear can be used to rotate a second optically trapped microgear clearly demonstrating how form birefringent microgears could be used to actuate a micromachine such as a microgear pump.

Laser induced heating at the interface between two immiscible fluids is used to produce thermocapillary stresses along this interface. When the interface is heated locally, the surface tension is reduced at the hot spot and the fluids are drawn in the direction opposite to the temperature gradient. This effect, known as the Marangoni effect, is amplified in miniaturized systems since the temperature and surface tension gradients are increased as the typical distances are reduced. When implemented in an adequate microchannel geometry, the Marangoni effect allows us to devise fundamental building blocks for microfluidic systems. In particular, the motion along the surface of a fluid immediately causes movement in the bulk for low Reynolds numbers, another feature of miniaturized systems. This allows us to apply the laser heating technique to make a pump in a microchannel, by focusing a laser beam on an oil-water interface. More surprisingly, localized heating can also be applied to create a microfluidic valve when implemented in an adequate geometry. A mixer can also be produced by using a fluid-fluid interface which changes in time. In this talk we present some of our realizations of these actuators in microfluidic channels and discuss some of the physical background underlying their behavior. Once these devices are created they can be combined in a complex circuit, yielding a contactless, scalable solution for the practical limitations that plague microfluidics.

Electro-osmotic pumping is an efficient way to move fluids in microfluidic systems. It is driven by the interaction of the Debye layer formed in the vicinity of the charged channel wall with a tangential electric field. The key parameters that determine the flow properties are the zeta potential of the surface and the electric field that drives the flow. Consequently, the flow can be controlled by appropriately modifying these parameters. Controlling the charge on the channel wall makes it possible to modify fluid flow. Likewise, the electric field close to the surface can be modified by changing the conductivity of the surface. The surface charge of appropriate materials can be changed by light illumination: the application of this phenomenon offers the possibility to optically control flow parameters. We have tested this possibility with several light sensitive surfaces. In the class of materials that change their charge upon illumination TiO₂, a well known photoactive material was investigated. Experiments were also performed with the protein bacteriorhodopsin, known to change its surface charge following the release of protons into the solvent upon illumination. CdS was tested as the photoconductive material to modify the electric field by light. Linear microfluidic channels were prepared by soft lithography: a PDMS mold was placed upon a planar glass surface so that a rectangular cross section channel was formed upon the glass. The photosensitive materials covered the bottom glass surface. The experiments show that the flow can be readily modulated by illumination. The results demonstrate that it is possible to dynamically control microfluidic flow, opening up the prospect to create optically controlled complex microfluidic networks.

In this article, we report the development of a novel system for distinguishing various types of cell based on the differences in the values of optical forces experienced by them. The laser induced optical forces arise when molecules comprising the cell interact with the photons of the laser beam resulting in an exchange of momentum. The basic principle is based on the phenomenon of laser cell guidance: a cell, in proximity of a weakly focused laser beam experiences a net force which attracts it to the centre of the beam and propels it along the axis of the beam. The 'guidance' is thus a direct result of the optical forces experienced by the cell. The guidance force experienced by the cell is directly related to the amount of momentum exchange, which in turn depends on factors such as the cell shape and size, the refractive index difference between the materials composing the cell and the materials in the culture media, and the properties of the beam. Since different cell types have different physical properties, they experience different optical (guiding) forces when they interact with the same laser beam. Since guidance force causes the cells to be propelled along the axis of the beam, the force with which the cell is propelled will directly determine the velocity with which the cell will move along the beam. Hence different types of cells will move with different velocities. Thus by studying this motion of cell, we in turn study the forces experienced by the cell. We propose that by analyzing the velocity with which cells are being guided by a laser beam, we can develop a technique to distinguish various cell types. Extensive GLMT theories calculate and predict theoretically the forces experienced by particles of a given size and refractive index for a given set of beam parameters. These theories are used as the basis to make judgments about the validity of our velocity and force calculation. The velocity assessments are conducted for a statistically significant number of cells from various cell types to explore the systematic difference among various groups of cells and, accordingly, the reliability of the developed method is discussed.

In the optical domain, the gradient force may be exploited in optical tweezers to confine high-index particles to points of maximum light intensity [1]. This methodology has enabled key advances in biology enabling a deeper understanding of molecular motors and the properties of DNA. Optical traps have also enabled a wide range of studies in optical angular momentum, colloid science and microfluidics. Recent work has shown that extended, optically tailored landscapes can offer a mechanism by which to arrange and accumulate microparticles in pre-described arrays [2]). The ability to sculpt and reconfigure the optical potential energy landscape external to the sample is a key component of such studies. We may add a tilt to the potential or, more generally, break symmetry, enabling unprecedented control over directed transport of particles [3] Three dimensional optical lattices may be used for sorting and fractionation of biological material in a microfluidic flow [4]. However it would be advantageous to be able to separate and even accumulate both biological and colloidal matter in the absence of any flow within any sample chamber. This would allow true compatibility of sorting and separation without the need to implement flows and microfluidic systems. We exploit the varying affinity of mesoscopic objects to a circularly symmetric optical landscape to demonstrate this effect and demonstrate separation of cells and chromosomes. The differing Kramers residence time in each part of the light pattern leads to a thermally activated method for sorting based on their hopping probabilities within the rings of the Bessel beam. Whilst we employ a Bessel beam to elucidate and demonstrate the dynamics of the sorting other tailored landscapes can also be used.

Microscopic particles with varying optical properties may be induced to move in different ways when placed on a sculpted optical potential due to differences in shape, size or polarisability. The separation of red blood cells (erythrocytes) and white blood cells (lymphocytes) is achieved in a non-invasive manner and in the absence of any microfluidic systems using a 'non-diffracting' circularly symmetric Bessel beam. The Bessel beam, which consists of a series of concentric rings, each of equal power and of 3.2μm thickness with a spacing of 2μm around a central maximum of 5μm diameter (and is akin to a rod of light as its propagation distance is 3mm), is directed upward into a sample chamber containing blood. Fluctuations in Brownian motion cause cells to escape from individual rings of the Bessel beam and travel towards the beam centre, where the intensity of the rings increases. However, these cells must be able to overcome the potential barrier of each ring which gets larger toward the central maximum. Lymphocytes - spherical in shape and 7μm in diameter (therefore overlapping two rings) - are transported, due to the gradient force of the optical field, to the beam centre where they are guided upwards and form a vertical stack, whereas erythrocytes re-align on their sides in the outer rings and are then guided upwards, because once aligned they cannot escape the potential barrier and 'lock-in' to that ring. The optical power required for optimal sorting in this static sorter which requires no fluid flow is investigated.

We describe a general way how to calculate optical forces and torque acting on colloids placed into laser interference field. In this paper we focus on a polystyrene particle placed into three interfering beams laying in one plane and creating two dimensional optical lattice. Colloids behavior in this type of optical landscape differs according to the colloid size. Spatial distribution of the optical force and torque is studied and particle behavior is predicted. Total optical force and torque are presented for various optical lattice configurations.

We have developed a method for measuring the red blood cell (RBC) membrane overall elasticity μ by measuring the deformation of the cells when dragged at a constant velocity through a plasma fluid by an optical tweezers. The deformability of erythrocytes is a critical determinant of blood flow in the microcirculation. We tested our method and hydrodynamic models, which included the presence of two walls, by measuring the RBC deformation as a function of drag velocity and of the distance to the walls. The capability and sensitivity of this method can be evaluated by its application to a variety of studies, such as, the measurement of RBC elasticity of sickle cell anemia patients comparing homozygous (HbSS), including patients taking hydroxyrea (HU) and heterozygous (HbAS) with normal donors and the RBC elasticity measurement of gamma irradiated stored blood for transfusion to immunosupressed patients as a function of time and dose. These studies show that the technique has the sensitivity to discriminate heterozygous and homozygous sickle cell anemia patients from normal donors and even follow the course of HU treatment of Homozygous patients. The gamma irradiation studies show that there is no significant change in RBC elasticity over time for up to 14 days of storage, regardless of whether the unit was irradiated or not, but there was a huge change in the measured elasticity for the RBC units stored for more than 21 days after irradiation. These finds are important for the assessment of stored irradiated RBC viability for transfusion purposes because the present protocol consider 28 storage days after irradiation as the limit for the RBC usage.

Granulocytes are a group of white blood cells belonging to the innate immune system in human and in murine in which eosinophils play an important role in worm infection-induced inflammation. The migration of these cells is well characterized and has been separated into four steps: rolling, adhesion, transendothelial migration, and chemotaxis, however, the physical characteristics of the chemotactic force to eosinophils from worm component remain largely unknown. Note that optical tweezers are featured in the manipulation of a single cell and the measurement of biological forces. Therefore, we propose to use optical tweezers to examine the chemotactic force to a eosinophil from a T. canis lavae preparation in terms of distance during the migration of eosinophil.

Following the recent advances in nano-optics, optical manipulation by evanescent fields instead of conventional propagating fields has recently awaken an increasing interest. The main advantages of using low dimensionality fields are (i) the possibility of integrating on a chip applications involving optical forces but also (ii) the absence of limitation by the diffraction limit for the trapping volume. Previous works have investigated theoretically and experimentally the guiding of dielectric and metallic beads at an interface sustaining an extended surface wave. In this work, we study theoretically the radiation forces exerted on Rayleigh dielectric beads under local evanescent illumination. Especially, we consider the configuration where a three-dimensional Gaussian beam is totally reflected at the interface of a glass prism. The results point out the illumination parameters where the gradient forces exceed the scattering force and allow for a stable trapping. The effect of the Goos-Haenchen shift on the location of the trapping site is also discussed.

Laser trapped mirrors may offer an interesting alternative for very large space telescopes. The use of sub-wavelength structures raises many problems concerning electromagnetism, solid state physics and out of equilibrium micro-scale thermodynamics. Optical trapping in low index media and damping in vaccum are among the main technical difficulties that must be addressed. New experimental techniques are explored. Theoretical issues are discussed, relating to Van der Waals cohesion energy and optical forces in the Mie and Rayleigh ranges.

We observe the orbital motion of particles in the Mie regime as they are trapped within the multiringed transverse field of a rotating Helical Mathieu light beam. The observed motion results as angular momentum is transferred to the particles from the orbital component of this novel light beam. Mathieu Beams present nondiffracting features within a limited distace are suitable for a variety of applications in optical manipulation where guiding and rotation of particles is required. Dynamic variables of the motion of the particles such as angular velocity and acceleration are measured and compared to the theoretical predictions from numerical simulation.

Cell sorting is important in quantitative analysis for cellular and molecular biology study. For this purpose, we develop an opto-electro-mechanical sorting system which integrates a holographic optical tweezers (HOT) system to a microfluidics device. This system is designed to manipulate and sort various micro objects of different sizes by an optical pattern which is dynamic and programmable. In this work, we fabricate a microchannel with a main channel and two branches with polydimethylsiloxane (PDMS) which is a biocompatible elastomer material by utilizing semiconductor micro-fabrication techniques. By using a HOT system, we also generate two linear optical traps for the manipulation of micro particles. As a result, we have demonstrated that the larger particles are guided to the second branch by the two linear optical traps while the smaller particles remain unaffected and flow into the first branch of the microchannel.

Recently, a new type of beam termed "spiral optical beam" has been introduced [Alonzo, et al., Opt. Express 13, 1749 (2005)]. Spiral beams are created from multiplicative mixtures of helical and conical phase distributions. Helico-conical phase fronts that generate these novel beams are not achieved with a sequence of a corkscrew wave-plate and an axicon (as this sequence gives a sum of helical and conical phase terms). Nevertheless, the availability of phase-only spatial light modulators (SLM) allows one to directly imprint helico-conical phase functions on an incident plane wave and provides an easy way to modify the profile of the encoded phase. Focusing the phase-modified field results in spiral intensity distributions that may find use for optical manipulation of mesoscopic particles. In this paper, we have extended the discussion to translation and rotation (as well as chirality switching) of the spiral beams using SLM control.

Higher-order laser beams were demonstrated to enable optical manipulation of low-index-particles. In this work single-ringed Laguerre-Gaussian beams, obtained by means of phase-only diffractive optical elements, are used to perform manipulation of phospholipid-shelled gas microbubbles in water. Implementation of diffractive optical elements on a programmable spatial light modulator allows to generate also arrays of Laguerre-Gaussian traps. We show manipulation of low-index particles by properly displaying a suitable sequence of diffractive optical elements. Control over the distance between the trapped particles in real time is also demonstrated.

Holographic tweezers offer a very versatile tool in many trapping applications. Compared to tweezers working with acousto optical modulators or using the generalized phase contrast, holographic tweezers so far were relatively slow. The computation time for a hologram was much longer than the modulation frequency of the modulator. To overcome this drawback we present a method using modified algorithms which run on state of the art graphics boards and not on the CPU. This gives the potential for a fast manipulation of many traps, for cell sorting for example, as well as for a real-time aberration control. The control of aberrations which can vary spatially or temporally is relevant to many real world applications. This can be accomplished by applying an iterative approach based on image processing.

There are increased needs for manipulating microscopic objects. One of enabling technologies is an instrument called optical tweezers (OT) that uses a focused laser beam to trap and move microscopic objects. OT has been shown effective for directly manipulating spherical, cylindrical or axis-symmetrical shapes. For other forms of shapes that do not show any symmetry, there have been works on using micrometer sized balls as a handle to indirectly manipulate the objects. Direct manipulation is difficult because complex trapping potential needs to be calculated to stably trap non-symmetrical shapes. User interfaces for these "indirect" systems use a computer mouse to design a layout of balls for surrounding (holding) an object and a trajectory that describes how these balls as a whole moves. The contained object pushed by these surrounding balls then moves accordingly. In this study, we introduce an intuitive user interface system for manipulating these balls. Using virtual reality gloves, each finger tip position of an operator is used to position control these balls. This user interface system enables the operator to intuitively grasp, move and release irregular formed shapes.

This report is concerned with investigations of real-time manipulation of polystyrene particles using a focused laser beam modulated in phase by dynamic holograms. The phase-only hologram generating in a computer-addressed spatial light modulator is calculated by the Gerheberg-Saxton algorithm to obtain the intensity pattern in the focal plane of an objective lens. The particles are trapped by a multi-spot array of the laser beam generated by the phase-only hologram and manipulated dynamically and continuously in real time.

The quantitative study of displacements and forces of motor proteins and processes that occur at the microscopic level and below require a high level of sensitivity. For optical traps, two techniques for position sensing have been accepted and used quite extensively: quadrant photodiodes and an interferometric position sensing technique based on DIC imaging. While quadrant photodiodes have been studied in depth and mathematically characterized, a mathematical characterization of the interferometric position sensor has not been presented to the authors' knowledge. The interferometric position sensing method works off of the DIC imaging capabilities of a microscope. Circularly polarized light is sent into the microscope and the Wollaston prism used for DIC imaging splits the beam into its orthogonal components, displacing them by a set distance determined by the user. The distance between the axes of the beams is set so the beams overlap at the specimen plane and effectively share the trapped microsphere. A second prism then recombines the light beams and the exiting laser light's polarization is measured and related to position. In this paper we outline the mathematical characterization of a microsphere suspended in an optical trap using a DIC position sensing method. The sensitivity of this mathematical model is then compared to the QPD model. The mathematical model of a microsphere in an optical trap can serve as a calibration curve for an experimental setup.

System identification methods are presented for the estimation of the characteristic frequency of an optically trapped particle. These methods are more amenable to automated on-line measurements and are believed to be less prone to erroneous results compared to techniques based on thermal noise analysis. Optical tweezers have been shown to be an effective tool in measuring the complex interactions of micro-scale particles with piconewton resolution. However, the accuracy of the measurements depends heavily on knowledge of the trap stiffness and the viscous drag coefficient for the trapped particle. The most commonly referenced approach to measuring the trap stiffness is the power spectrum method, which provides the characteristic frequency for the trap based on the roll-off of the frequency response of a trapped particle excited by thermal fluctuations. However, the reliance on thermal fluctuations to excite the trapping dynamics results in a large degree of uncertainty in the estimated characteristic frequency. These issues are addressed by two parameter estimation methods which can be implemented on-line for fast trap characterization. The first is a frequency domain system identification approach which combines swept-sine frequency testing with a least-squares transfer function fitting algorithm. The second is a recursive least-squares parameter estimation scheme. The algorithms and results from simulation studies are discussed in detail.

In this report, we describe a noninvasive methodology for manipulating single Mb-size whole-genome DNA molecules. Cells were subjected to osmotic shock and the genome DNA released from the burst cells was transferred to a region of higher salt concentration between cover slips using optical tweezers. The transferred genome DNA exhibits a conformational transition from a compact state into an elongated state, accompanied by the change in its environment. Here, the applicability of optical tweezers to the on-site manipulation of giant genomic DNA is suggested. Next, to control the field environment more precisely, a flow chamber was made and similar investigations were carried out. In the flow chamber, the higher-order structure of individual chromosomal DNA molecules from a fission yeast that were folded by polyamine was changed to a partially unfolded form by transporting into a higher salt condition using optical tweezers. These promising methodologies demonstrated here may make it possible to recover an intact single whole-genome DNA from a cell and carry out further sequential investigations under a microscope.

The displacements of a polystyrene microsphere trapped by an optical tweezers (OT) can be used as a force transducer for mechanical measurements in life sciences such as the measurement of forces of living microorganisms or the viscosity of local fluids. The technique we used allowed us to measure forces on the 200 femto Newtons to 4 pico Newtons range of the protozoa Leishmania amazonensis, responsible for a serious tropical disease. These observations can be used to understand the infection mechanism and chemotaxis of these parasites. The same technique was used to measure viscosities of few microliters sample with agreement with known samples better than 5%. To calibrate the force as a function of the microsphere displacement we first dragged the microsphere in a fluid at known velocity for a broad range of different optical and hydrodynamical parameters. The hydrodynamical model took into account the presence of two walls and the force depends on drag velocity, fluid viscosity and walls proximities, while the optical model in the geometric optics regime depends on the particle and fluid refractive indexes and laser power. To measure the high numerical (NA) aperture laser beam power after the objective we used an integration sphere to avoid the systematic errors of usual power meters for high NA beams. After this careful laser power measurement we obtained an almost 45 degrees straight line for the plot of the optical force (calculated by the particle horizontal displacement) versus hydrodynamic force (calculated by the drag velocity) under variation of all the parameters described below. This means that hydrodynamic models can be used to calibrate optical forces, as we have done for the parasite force measurement, or vice-versa, as we did for the viscosity measurements.

The purpose of this study is to seek a correlation between the swimming forces of sperm, their swimming speed and the speed of progression score (SOP), which is given to them by the generally applied subjective 1 - 5 system. This study also examines the effects of length of exposure to the laser trap and laser power on sperm motility. Sperm with SOPs of 2-4 were trapped by a continuous wave 1064 nm single point gradient laser trap. To study trap duration effects, sperm were trapped for fifteen, ten and five seconds at 420mW laser power. To study laser power effects within the trap, powers of 420mW, 350mW, 300mW, and 250mW were applied for a constant duration (5 seconds). The correlations between sperm swimming force, swimming speed and speed of progression were examined at a trap duration and trapping power that were statistically shown to not significantly affect sperm motility. Swimming forces were measured by trapping sperm and subsequently decreasing laser power until the sperm were capable of escaping the trap (escape laser power is directly proportional to swimming force). Swimming speeds were calculated by custom built software, and speed of progression scores were assigned in a blind study by fertility experts examining offline video of the experiments. Sperm swimming force measurements by optical trapping may be a method to quantitatively measure sperm vitality that augments currently used methods.

We present an alternative approach to image and analyze a large amount of optically-bound particles without using microscope objective lenses. The polystyrene spheres were trapped and patterned in the one dimensional optical potential traps formed by two counter-propagating laser beams. Diffraction patterns from such polystyrene sphere assembly were studied and the result are reported. This method offers a significantly larger field of view and faster analysis over the conventional high-power microscope-based imaging technique. The applications of such technique to investigate the statistic (macroscopic) behaviors of a large number of microscopic particles trapped in the optical field were proposed.

We focus on a numerical study using coupled dipoles to explore the interaction between two spheres of micron size. This interaction (sometimes called optical coupling or binding) is studied in counter-propagating Gaussian laser beams that do not interfere. We found that for a certain range of the refractive indices of the particle there exist several stable and unstable positions where the total forces acting on particles are zero. Moreover we observed an oscillations of the force acting between both objects coming from the interference of backscattered field.

Recently, emerging from studies based on quantum electrodynamics, it has been shown possible to significantly modify the form and magnitude of inter-particle forces by the throughput of intense laser light. This paper identifies these laser-induced forces as being the result of coherent stimulated scattering by particle pairs. Such forces have the capacity to generate novel patterns of nanoscale response, entirely determined and controlled by the frequency, intensity, polarisation and other features of the laser input. Results are given, based on general calculations of the optical forces and torques operating between a pair of dielectric particles. It is subsequently shown, by further development of the analysis, that it is possible to address the case of a twisted (Laguerre-Gaussian) laser beam as the input radiation. Here, the results reveal additional and highly distinctive torques operating between pairs of nanoparticles. Significantly the results demonstrate that these laser-induced forces and torques can be either positive or negative according to conditions. As a consequence, new possibilities emerge for the optical control of nanoparticle ordering, clustering and trapping.

Here we report on the motion of microscopic, optically trapped glass rods suspended in water and experiencing a light torque. The motion consists of two distinct regimes: a linear regime where the rod angle increases linearly with time and a nonlinear regime where the rod angle changes nonlinearly, experiencing accelerations and rapid reversals. These regimes depend on whether the rotation frequency of the linearly polarized driving light is above or below a critical frequency, Ω_c. We will present experimental data that spans both regimes. We will also provide a theoretical model that agrees with the observed motion. We are working on extending this effort on the optical trapping and rotation of rods to smaller scales, where the diameter is 100 nm or less. This scaling down will allow us to study the nonlinear motion near a surface. Such studies can help us to understand surface effects that are important in micro- and nanofluidics. Toward this end, we report results on our attempts to trap silver nanorods of diameter close to 100 nm suspended in acetone.

One of the main problems in using oil immersed objectives for optical trapping is due to the reflective index mismatch. While in water immersed lens or in "classical" ones the index mismatch is, at most, because of the propagation from a low-index to a high-index medium so no big aberrations appear, in the oil-immersed ones the aberrations cannot be neglected. Due to the propagation of a focalized beam from a high refraction index medium to a low one, spherical aberrations appear. But the numerical aperture an oil immersed objective can reach is usually above 1.2, so theoretically, the Q-factor can reach higher values. In this paper we confront two different methods of force calculations. The first one is based on Ashkin's (Biophys. J. - Feb. 1992) formulas and uses a ray-tracing approach. Using this approach we can observe the asymmetry of the forces along the optical axis and the high Q-factors values on the orthogonal plane. The second approach is a more rigorous one and is based on wave optics. We analyze the Gaussian beam propagation using the scalar version of the diffraction theory and the formulas of the scattering and gradient forces developed by W.H.Wright &al. (Applied Optics - March 1994). This approach, although more precise in defining the focus shape, is not necessarily more precise in determining the force value. This essentially because of the approximations used in the force formulas. While in the ray-tracing approach, the error can be minimized by changing the sampling period of the beam, in the wave-optics approach this cannot be done. In the same time, in the latter approach, the beam shape can be described better so the accuracy of the simulation improved. To our belief the possibility of using both simulations must be taken into account while in the "resonance" regime and, function of the needs one has, decide which one is to be considered reliable.

There are basically two types of high-resolution spatial light modulators (SLMs): reflective and transmissive. Obviously, the two types lend themselves to different optical configurations, where one might have advantages over the other for generating the dynamic multi-spot patterns needed by the micromanipulator. In addition, there are inherent performance and operational differences between the two, which has several implications. This paper compares reflective and transmissive high resolution SLMs as well as optical and electrical addressing schemes for operation in optical manipulation applications.

Sperm motility is an important area in understanding male infertility. Various techniques, such as the Computer Assisted Sperm Analysis (CASA), have been used to understand sperm motility. Sperm motility is related to the energy (ATP) production of sperm. ATP is produced by the depolarization of the membrane potential of the inner membrane of the mitochondria. In this study, a mitochondrial dye, JC-1, has been used to monitor the energetics of the mitochondria. This fluorescent dye can emit at two different wavelengths, depending on the membrane potential of the mitochondria. It can fluoresce green at low membrane potential and red at high membrane potential. The ratio of the two colors (red/green) allows for an accurate measurement of the change of membrane potential. Various experiments were conducted to quantify the behavior of the dye within the sperm and the reaction of the sperm to trap. Sperm were trapped using laser tweezers. Results have shown that the ratio drops dramatically when sperm are trapped, indicating a depolarization of the membrane. The physiological response to this depolarization is yet to be determined, but the studies indicate that the sperm could have been slightly damaged by the laser. However, knowing that sperm depolarizes their membrane when trapped can help understand how sperm react to their environment and consequently help treat male infertility.