Parallel analysis of individual biological cells using multifocal laser tweezers Raman spectroscopy

We report on the development and characterization of a multifocal laser tweezers Raman spectroscopy (M-LTRS) technique for parallel Raman spectral acquisition of individual biological cells. Using a 785-nm diode laser and a time-sharing laser trapping scheme, multiple laser foci are generated to optically trap single polystyrene beads and suspension cells in a linear pattern. Raman signals from the trapped objects are simultaneously projected through the slit of a spectrometer and spatially resolved on a charge-coupled device (CCD) detector with minimal signal crosstalk between neighboring cells. By improving the rate of single-cell analysis, M-LTRS is expected to be a valuable method for studying single-cell dynamics of cell populations and for the development of high-throughput Raman based cytometers.     

Optical trapping of fluorescent beads

Optical tweezers have been successfully used to trap a variety of particles and biological specimens for numerous applications. Particles which are reflective as well as absorbing could be trapped using beams such as optical vortex. Here we give the details of our efforts to trap fluorescent microparticles. We have set up an optical trap for these fluorescent microparticles using holographic optical tweezers; we observe that it is not possible to trap fluorescent microparticles with a Gaussian laser beam or a hollow beam. However, as the fluorescence of these particles gets degraded they could be trapped in custom-made holographic tweezers. Moreover, when a fluorescent particle is brought in the trap containing stably trapped non-fluorescent particle, the stably trapped non-fluorescent particle also escapes from the trap.     

Sorting of micron-sized particles using holographic optical Raman tweezers in aqueous medium

We have constructed a holographic optical tweezers system combined with Raman spectroscopy to sort trapped particles. Our software automatically moves the trapped objects to the measurement positions to obtain individual Raman signals from multiple trapped particles. We performed the sorting by comparing their spectra with the previously measured training dataset using the correlation coefficients. We used yeast cells and polystyrene beads as test particles. This study aims to show that biological particles can be separated using single cell analysis with combined holographic optical tweezers and Raman spectroscopy system.     

Using polarization-shaped optical vortex traps for single-cell nanosurgery

Single-cell nanosurgery and the ability to manipulate nanometer-sized subcellular structures with optical tweezers has widespread applications in biology but so far has been limited by difficulties in maintaining the functionality of the transported subcellular organelles. This difficulty arises because of the propensity of optical tweezers to photodamage the trapped object. To address this issue, this paper describes the use of a polarization-shaped optical vortex trap, which exerts less photodamage on the trapped particle than conventional optical tweezers, for carrying out single-cell nanosurgical procedures. This method is also anticipated to find broad use in the trapping of any nanoparticles that are adversely affected by high-intensity laser light.     

Mapping the restoring force of an optical trap in three dimensions using a two laser dual-trap approach

A two laser optical tweezers set-up is developed and used to measure deflections of a microsphere trapped in a calibrated spatial light modulator steered probe trap as it is stepped through a three dimensional grid about a fixed test trap. These measurements are used to map the restoring force of the test trap on the microsphere in three dimensions. Results are validated over a common range by comparison to drag force measurements for both silica and polystyrene microspheres.     

Laser Heating Tunability by Off‐Resonant Irradiation of Gold Nanoparticles

Temperature changes in the vicinity of a single absorptive nanostructure caused by local heating have strong implications in technologies such as integrated electronics or biomedicine. Herein, the temperature changes in the vicinity of a single optically trapped spherical Au nanoparticle encapsulated in a thermo‐responsive poly(N‐isopropylacrylamide) shell (Au@pNIPAM) are studied in detail. Individual beads are trapped in a counter‐propagating optical tweezers setup at various laser powers, which allows the overall particle size to be tuned through the phase transition of the thermo‐responsive shell. The experimentally obtained sizes measured at different irradiation powers are compared with average size values obtained by dynamic light scattering (DLS) from an ensemble of beads at different temperatures. The size range and the tendency to shrink upon increasing the laser power in the optical trap or by increasing the temperature for DLS agree with reasonable accuracy for both approaches. Discrepancies are evaluated by means of simple models accounting for variations in the thermal conductivity of the polymer, the viscosity of the aqueous solution and the absorption cross section of the coated Au nanoparticle. These results show that these parameters must be taken into account when considering local laser heating experiments in aqueous solution at the nanoscale. Analysis of the stability of the Au@pNIPAM particles in the trap is also theoretically carried out for different particle sizes.     

Optical manipulation in combination with multiphoton microscopy for single-cell studies

We demonstrate how optical tweezers can be incorporated into a multiphoton microscope to achieve three-dimensional imaging of trapped cells. The optical tweezers, formed by a cw 1064 nm Nd:YVO4 laser, were used to trap live yeast cells in suspension while the 4',6-diamidino-2-phenylindole-stained nucleus was imaged in three dimensions by use of a pulsed femtosecond laser. The trapped cell was moved in the axial direction by changing the position of an external lens, which was used to control the divergence of the trapping laser beam. This gives us a simple method to use optical tweezers in the laser scanning of confocal and multiphoton microscopes. It is further shown that the same femtosecond laser as used for the multiphoton imaging could also be used as laser scissors, allowing us to drill holes in the membrane of trapped spermatozoa.     

Axial optical trapping forces on two particles trapped simultaneously by optical tweezers

Optical tweezers, which utilize radiation pressure to control and manipulate microscopic particles, are used for a large number of applications in biology and colloid science. In most applications a single optical tweezers is used to control one single particle. However, two or more particles can be trapped simultaneously. Although this characteristic has been used in applications, no theoretical analysis of the trapping force or the status of the trapped particles is available to our knowledge. We present our calculation, using a ray optics model, of the axial trapping forces on two rigid particles trapped in optical tweezers. The spherical aberration that results from a mismatch of the refractive indices of oil and water is also considered. The results show that the forces exerted by the optical tweezers on the two particles will cause the two particles to touch each other, and the two particles can be stably trapped at a joint equilibrium point. We also discuss the stability of axial trapping. The calculation will be useful in applications of optical tweezers to trap multiple particles.     

Spatially resolved analysis of small particles by confocal Raman microscopy: depth profiling and optical trapping

Raman microscopy is a powerful method to provide spatially resolved information about the chemical composition of materials. With confocal collection optics, the method is well suited to the analysis of small particles, either resting on a surface or optically trapped at a laser focus, where the confocal collection volume optimizes the signal from the particle. In this work, the sensitivity and spatial selectivity of detecting Raman scattering from single particles was determined as a function of particle size. An inverted confocal Raman microscope was used to acquire spectra of individual surface-bound and optically trapped polystyrene particles with sizes ranging between 200 nm and 10 microm. The particles are in contact with aqueous solution containing perchlorate ion that served as a solution-phase Raman-active probe to detect interferences from the surrounding medium. The collection volume is scanned through single particles that are attached to the surface of the coverslip, and the sensitivity and selectivity of detection are measured versus particle size. The results compare favorably with a theoretical analysis of the excitation profile and confocal collection efficiency integrated over the volumes of the spherical particles and the surrounding solution. This analysis was also applied to the detection of particles that are optically trapped and levitated above the surface of the coverslip. The results are consistent with the optical trapping of particles at or near the excitation beam focus, which optimizes excitation and selective collection of Raman scattering from the particle.     

Rapid rotation of micron and submicron dielectric particles measured using optical tweezers

Abstract

We demonstrate the use of a laser trap (‘optical tweezers’) and back-focal-plane position detector to measure rapid rotation in aqueous solution of single particles with sizes in the vicinity of 1 μm. Two types of rotation were measured: electrorotation of polystyrene microspheres and rotation of the flagellar motor of the bacterium Vibrio alginolyticus. In both cases, speeds in excess of 1000 Hz (rev s^?1) were measured. Polystyrene beads of diameter about 1 μm labelled with smaller beads were held at the centre of a microelectrode array by the optical tweezers. Electrorotation of the labelled beads was induced by applying a rotating electric field to the solution using microelectrodes. Electrorotation spectra were obtained by varying the frequency of the applied field and analysed to obtain the surface conductance of the beads. Single cells of V. alginolyticus were trapped and rotation of the polar sodium-driven flagellar motor was measured. Cells rotated more rapidly in media containing higher concentrations of Na⁺, and photodamage caused by the trap was considerably less when the suspending medium did not contain oxygen. The technique allows single-speed measurements to be made in less than a second and separate particles can be measured at a rate of several per minute.     

Reagentless identification of single bacterial spores in aqueous solution by confocal laser tweezers Raman spectroscopy

We demonstrate that optical trapping combined with confocal Raman spectroscopy using a single laser source is a powerful tool for the rapid identification of micrometer-sized particles in an aqueous environment. Optical trapping immobilizes the particle while maintaining it in the center of the laser beam path and within the laser focus, thus maximizing the collection of its Raman signals. The single particle is completely isolated from other particles and substrate surfaces, therefore eliminating any unwanted background signals and ensuring that information is collected only from the selected, individual particle. In this work, an inverted confocal Raman microscope is combined with optical trapping to probe and analyze bacterial spores in solution. Rapid, reagentless detection and identification of bacterial spores with no false positives from a complex mixed sample containing polystyrene and silica beads in aqueous suspension is demonstrated. In addition, the technique is used to analyze the relative concentration of each type of particle in the mixture. Our results show the feasibility for incorporating this technique in combination with a flow cytometric-type scheme in which the intrinsic Raman signatures of the particles are used instead of or in addition to fluorescent labels to identify cells, bacteria, and particles in a wide range of applications.     

Measuring microscopic viscosity with optical tweezers as a confocal probe

We demonstrate, what is to the best our knowledge, a new method for studying the motion of a particle trapped by optical tweezers; in this method the trapping beam itself is used as a confocal probe. By studying the response of the particle to periodic motion of the tweezers, we obtain information about the medium viscosity, particle properties, and trap stiffness. We develop the mathematical model, demonstrate experimentally its validity for our system, and discuss advantages of using this method as a new form of scanning photonic force microscopy for applications in which a high spatial and temporal resolution of the medium viscosity is desired.     

Three-dimensional force calibration of optical tweezers

Abstract

We demonstrate a method for 3-dimensional force calibration of optical tweezers by recording the trapping dynamics of polystyrene beads. This is realized by time-resolved detection of the horizontal and vertical position of a bead which is drawn to the focus of a laser beam. The method provides real time characterization of the force profile of an optical trap in all directions.     

Optimization of probe-laser focal offsets for single-particle tracking

In optical tweezers applications, tracking a trapped particle is essential for force measurement. One of the most popular techniques for single-particle tracking is achieved by analyzing the forward and backward light pattern, scattered by the target particle trapped by a trap laser beam, of an additional probe-laser beam with different wavelength whose focus is slightly apart from the trapping center. However, the optimized focal offset has never been discussed. In this paper, we investigate the tracking range and sensitivity as a function of the focal offset between the trapping and the probe-laser beams. As a result, the optimized focal offsets are a 3.3-fold radius ahead and a 2.0-fold radius behind the trapping laser focus in the forward tracking and the backward tracking, respectively. The experimental result agrees well with a theoretical prediction using the Mie scattering theory.     

High trapping forces for high-refractive index particles trapped in dynamic arrays of counterpropagating optical tweezers

We demonstrate the simultaneous trapping of multiple high-refractive index (n > 2) particles in a dynamic array of counterpropagating optical tweezers in which the destabilizing scattering forces are canceled. These particles cannot be trapped in single-beam optical tweezers. The combined use of two opposing high-numerical aperture objectives and micrometer-sized high-index titania particles yields an at least threefold increase in both axial and radial trap stiffness compared to silica particles under the same conditions. The stiffness in the radial direction is obtained from measured power spectra; calculations are given for both the radial and the axial force components, taking spherical aberrations into account. A pair of acousto-optic deflectors allows for fast, computer-controlled manipulation of the individual trapping positions in a plane, while the method used to create the patterns ensures the possibility of arbitrarily chosen configurations. The manipulation of high-index particles finds its application in, e.g., creating defects in colloidal photonic crystals and in exerting high forces with low laser power in, for example, biophysical experiments.     

Quantitative optical trapping of single gold nanorods

We report a quantitative analysis of the forces acting on optically trapped single gold nanorods. Individual nanorods with diameters between 8 and 44 nm and aspect ratios between 1.7 and 5.6 were stably trapped in three dimensions using a laser wavelength exceeding their plasmon resonance wavelengths. The interaction between the electromagnetic field of an optical trap and a single gold nanorod correlated with particle polarizability, which is a function of both particle volume and aspect ratio.     

Single-particle mass spectrometry of polystyrene microspheres and diamond nanocrystals.

High-resolution mass spectra of single submicrometer-sized particles are obtained using an electrospray ionization source in combination with an audio frequency quadrupole ion-trap mass spectrometer. Distinct from conventional methods, light scattering from a continuous Ar-ion laser is detected for particles ejected out of the ion trap. Typically, 10 particles are being trapped and interrogated in each measurement. With the audio frequency ion trap operated in a mass-selective instability mode, analysis of the particles reveals that they all differ in mass-to-charge ratio (m/z), and the individual peak in the observed mass spectrum is essentially derived from one single particle. A histogram of the spectra acquired in 10(2) repetitions of the experiment is equivalent to the single spectrum that would be observed when an ion ensemble of 10(3) particles is analyzed simultaneously using the single-particle mass spectrometer (SPMS). To calibrate such single-particle mass spectra, secular frequencies of the oscillatory motions of the individual particle within the trap are measured, and the trap parameter qz at the point of ejection is determined. A mass resolution exceeding 10(4) can readily be achieved in the absence of ion ensemble effect. We demonstrate in this work that the SPMS not only allows investigations of monodisperse polystyrene microspheres, but also is capable of detecting diamond nanoparticles with a nominal diameter of 100 nm, as well.     

Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy

We report on a rapid method for reagentless identification and discrimination of single bacterial cells in aqueous solutions using a combination of laser tweezers and confocal Raman spectroscopy (LTRS). The optical trapping enables capturing of individual bacteria in aqueous solution in the focus of the laser beam and levitating the captured cell well off the cover plate, thus maximizing the excitation and collection of Raman scattering from the cell and minimizing the unwanted background from the cover plate and environment. Raman spectral patterns excited by a near-infrared laser beam provide intrinsic molecular information for reagentless analysis of the optically isolated bacterium. In our experiments, six species of bacteria were used to demonstrate the capability of the confocal LTRS in the identification and discrimination between the diverse bacterial species at various growth conditions. We show that synchronized bacterial cells can be well-discriminated among the six species using principal component analyses (PCA). Unsynchronized bacterial cells that are cultured at stationary phases can also be well-discriminated by the PCA, as well as by a hierarchical cluster analysis (HCA) of their Raman spectra. We also show that unsynchronized bacteria selected from random growth phases can be classified with the help of a generalized discriminant analysis (GDA). These findings demonstrate that the LTRS may find valuable applications in rapid sensing of microbial cells in diverse aqueous media.     

Optical tweezers: 20 years on

In 1986, Arthur Ashkin and colleagues published a seminal paper in Optics Letters, 'Observation of a single-beam gradient force optical trap for dielectric particles' which outlined a technique for trapping micrometre-sized dielectric particles using a focused laser beam, a technology which is now termed optical tweezers. This paper will provide a background in optical manipulation technologies and an overview of the applications of optical tweezers. It contains some recent work on the optical manipulation of aerosols and concludes with a critical discussion of where the future might lead this maturing technology.     

Hydrodynamic tweezers: 1. Noncontact trapping of single cells using steady streaming microeddies

A key need for dynamic single-cell measurements is the ability to gently position cells for repeated measurements without perturbing their behavior. We describe a new method that uses a gentle secondary flow to trap and suspend single cells, including motile cells, at predictable locations in 3-D. Trapped cells can be more dense or less dense than the surrounding medium. The cells are suspended without surface contact in one of four steady streaming eddies created by audible-frequency fluid oscillation (< or =1000 Hz) in a microchannel containing a single fixed cylinder (radius = 125 microm). Comparison of measured trap locations to computations of the eddy flow show that each trap is located near the eddy center, and the location is controlled via the oscillation frequency. We use the motile phytoplankton cell (Prorocentrum micans) to experimentally measure the trapping force, which is controlled via the oscillation amplitude. Trapping forces up to 30 pN are generated while exerting moderate shear stresses (shear stresses < or = 1.5 N/m2) on the trapped cell. The magnitude of this trapping force is comparable to that of optical tweezers or dielectrophoretic traps, without requiring an external field outside the physiological range for cells (the shear stresses are comparable to those found in arterial blood flow). The unique combination of predictable 3-D positioning, insensitivity to cell and medium properties, strong adjustable trapping forces, and a gentle fluid environment makes hydrodynamic tweezers a promising new option for noncontact trapping of single cells in suspension.