Identification of Protein Domains on Major Pilin MrkA that Affects Mechanical Properties of Klebsiella pneumoniae Type 3 Fimbriae

Chia-Han Chan, FENG-JUNG CHEN, Ying-Jung Huang, Shin-Yu Chen, Kuo-Liang Liu, Zhe-Chong Wang, Hwei-Ling Peng, Tri-Rung Yew, Cheng-Hsien Liu, Gan-Guang Liou, Ken Y. Hsu, Hwan-You Chang, and Long Hsu

The Klebsiella pneumoniae type 3 fimbriae are mainly composed of MrkA pilins that assemble into a helix-like filament. This study determined the biomechanical properties of the fimbriae and analyzed 11 site-directed MrkA mutants to identify domains critical for the properties. Escherichia coli strains expressing type 3 fimbriae with an Ala substitution either at F34, V45, C87, G189, T196, or Y197 resulted in a significant reduction in biofilm formation. The E. coli strain expressing MrkAG189A remained capable of producing a normal amount of fimbriae. Although F34A, V45A, T196A, and Y197A substitutions expressed on E. coli strains produced sparse quantities of fimbriae, no fimbriae were observed on the cells expressing MrkAC87A. Further investigations of the mechanical properties of the MrkAG189A fimbriae with optical tweezers revealed that, unlike the wild-type fimbriae, the uncoiling force for MrkAG189A fimbriae was not constant. The MrkAG189A fimbriae also exhibited a lower enthalpy in the differential scanning calorimetry analysis. Together, these findings indicate that the mutant fimbriae are less stable than the wild type. In conclusion, this study has demonstrated that the C-terminal β-strands of MrkA are required for the assembly and structural stability of fimbriae.

DOI

Optical Funneling and Trapping of Gold Colloids in Convergent Laser Beams

Andreas Koeniger and Werner Koehler

The simultaneous trapping of a large number of sedimenting Au colloids by optical radiation forces has been studied in detail. The particles are collected by a convergent laser beam and compressed against gravity and osmotic pressure at the upper window of the cell, thereby forming a dense colloidal gas. A minimum critical laser power is required to transport colloids into the trap. In contrast to conventional optical tweezers, the trap cannot be described by a 3d-potential. Once the trap is sufficiently filled, the laser power can be reduced below the critical value, thereby stabilizing the trap population. Some characteristic properties of the trap, like the critical laser power and the transit time, are readily understood from a simple deterministic model. A detailed description that is capable of quantitatively accounting for the time dependence of the trap population, the finite leak rate at low power levels, or hysteresis effects requires the incorporation of fluctuations by means of a proper Langevin equation. Multiple independent traps are realized by time multiplexing of the laser beam, which allows for splitting up, independent manipulation, and subsequent recombination of a trapped colloidal cloud.

DOI

Optical forces in plasmonic nanoantennas

A S Shalin and S V Sukhov

The optical forces acting on nanoparticles in V-shaped plasmonic resonators with a high local-field gain have been investigated. Two versions are considered, which make it possible to implement either attractive or repulsive gradient optical forces. A plasmonic resonator is proposed, which can focus 350-nm radiation and implement a repulsive gradient force. It has been shown for the first time that a perturbation induced by a nanoparticle redistributes the field in the resonator so that additional intensity peaks arise in both versions to hold the nanoparticle in the resonator by forming an optical trap.

DOI

Behavior of double emulsions in a cross-type optical separation system

Kyung Heon Lee, Sang Bok Kim, Sang Youl Yoon, Kang Soo Lee, Jin Ho Jung, and Hyung Jin Sung

The behavior of double emulsions in a cross-type optical particle separation system was studied for different combinations of the refractive indices and different inner and outer layer radii. The radii and refractive indices of the double emulsions were easily adjusted by taking advantage of the co-flowing geometry of a cross-type optical particle separation device. An analytic expression of the optical forces on a pair of concentric spheres was derived using the photon stream method in the ray optics regime. The predicted trajectories of the double emulsions by the optical force agreed well with the experimental data. This work has potential uses in cell separation by morphometry, drug delivery vehicle and emulsion-based biomedical applications.

DOI

Role of interfering optical fields in the trapping and melting of gold nanorods and related clusters

Hai-Dong Deng, Guang-Can Li, Qiao-Feng Dai, Min Ouyang, Sheng Lan, Achanta Venu Gopal, Vyacheslav A. Trofimov, and Tatiana M. Lysak

We investigate the simultaneous trapping and melting of a large number of gold (Au) nanorods by using a single focused laser beam at 800 nm which is in resonance with the longitudinal surface plasmon resonance of Au nanorods. The trapping and melting processes were monitored by the two-photon luminescence of Au nanorods. A multi-ring-shaped pattern was observed in the steady state of the trapping process. In addition, optical trapping of clusters of Au nanorods in the orbits circling the focus was observed. The morphology of the structure after trapping and melting of Au nanorods was characterized by scanning electron microscope. It was revealed that Au nanorods were selectively melted in the trapping region. While Au nanorods distributed in the dark rings were completely melted, those located in the bright rings remain unmelted. The multi-ring-shaped pattern formed by the interference between the incident light and the scattered light plays an important role in the trapping and melting of Au nanorods.

DOI

Development of a dual joystick-controlled laser trapping and cutting system for optical micromanipulation of chromosomes inside living cells

Marcellinus S. Harsono, Qingyuan Zhu, Linda Z. Shi, Michelle Duquette, Michael W. Berns

A multi-joystick robotic laser microscope system used to control two optical traps (tweezers) and one laser scissors has been developed for subcellular organelle manipulation. The use of joysticks has provided a “user-friendly” method for both trapping and cutting of organelles such as chromosomes in live cells. This innovative design has enabled the clean severing of chromosome arms using the laser scissors as well as the ability to easily hold and pull the severed arm using the laser tweezers.

DOI

Optical Manipulation of Liquid Crystal Droplets Through Holographic Polarized Tweezers: Magnus Effect

J. Hernández, C. Provenzano, P. Pagliusi & G. Cipparrone

We investigate the manipulation and trapping features of polarization holographic tweezers with liquid-crystal (LC) droplets in water. The force related to light's polarization pattern translates isotropic particles, while for birefringent particles exert forces and torques in opposite directions depending on the position of the particle. Experiments with LC emulsions allow verify expected scenarios and observe unconventional trapping of spinning birefringent particles in circularly-polarized fringes. This unusual trapping featured by rotating-bipolar-droplets suggest the involvement of one hydrodynamic force or Magnus Effect at low Reynolds number. This result shows that Magnus force contributes to opto-hydrodynamic trapping and is demonstrated with an experiment.

DOI

Analysis of the variation in the determination of the shear modulus of the erythrocyte membrane: Effects of the constitutive law and membrane modeling

P. Dimitrakopoulos

Despite research spanning several decades, the exact value of the shear modulus Gs of the erythrocyte membrane is still ambiguous, and a wealth of studies, using measurements based on micropipette aspirations, ektacytometry systems and other flow chambers, and optical tweezers, as well as application of several models, have found different average values in the range 2–10 μN/m. Our study shows that different methodologies have predicted the correct shear modulus for the specific membrane modeling employed, i.e., the variation in the shear modulus determination results from the specific membrane modeling. Available experimental findings from ektacytometry systems and optical tweezers suggest that the dynamics of the erythrocyte membrane is strain hardening at both moderate and large deformations. Thus the erythrocyte shear modulus cannot be determined accurately using strain-softening models (such as the neo-Hookean and Evans laws) or strain-softening/strain-hardening models (such as the Yeoh law), which overestimate the erythrocyte shear modulus. According to our analysis, the only available strain-hardening constitutive law, the Skalak et al. law, is able to match well both deformation-shear rate data from ektacytometry and force-extension data from optical tweezers at moderate and large strains, using an average value of the shear modulus of Gs=2.4–2.75 μN/m, i.e., very close to that found in the linear regime of deformations via force-extension data from optical tweezers, Gs=2.5±0.4 μN/m. In addition, our analysis suggests that a standard deviation in Gs of 0.4–0.5 μN/m (owing to the inherent differences between erythrocytes within a large population) describes well the findings from optical tweezers at small and large strains as well as from micropipette aspirations.

DOI

Optical Forces and Torques in Nonuniform Beams of Light

David B. Ruffner and David G. Grier

The spin angular momentum in an elliptically polarized beam of light plays several noteworthy roles in optical traps. It contributes to the linear momentum density in a nonuniform beam, and thus to the radiation pressure exerted on illuminated objects. It can be converted into orbital angular momentum, and thus can exert torques even on optically isotropic objects. Its curl, moreover, contributes to both forces and torques without spin-to-orbit conversion. We demonstrate these effects experimentally by tracking colloidal spheres diffusing in elliptically polarized optical tweezers. Clusters of spheres circulate deterministically about the beam’s axis. A single sphere, by contrast, undergoes stochastic Brownian vortex circulation that maps out the optical force field.

DOI

Tunable optical tweezers for wavelength-dependent measurements

Brooke Hester, Gretchen K. Campbell, Carlos López-Mariscal, Carly Levin Filgueira, Ryan Huschka, Naomi J. Halas, and Kristian Helmerson

Optical trapping forces depend on the difference between the trap wavelength and the extinction resonances of trapped particles. This leads to a wavelength-dependent trapping force, which should allow for the optimization of optical tweezers systems, simply by choosing the best trapping wavelength for a given application. Here we present an optical tweezer system with wavelength tunability, for the study of resonance effects. With this system, the optical trap stiffness is measured for single trapped particles that exhibit either single or multiple extinction resonances. We include discussions of wavelength-dependent effects, such as changes in temperature, and how to measure them.

DOI

Thermally activated state transition technique for femto-Newton-level force measurement

Feng-Jung Chen, Jhih-Sian Wong, Ken Y. Hsu, and Long Hsu

We develop and test a thermally activated state transition technique for ultraweak force measurement. As a force sensor, the technique was demonstrated on a classical Brownian bead immersed in water and restrained by a bistable optical trap. A femto-Newton-level flow force imposed on this sensor was measured by monitoring changes in the transition rates of the bead hopping between two energy states. The treatment of thermal disturbances as a requirement instead of a limiting factor is the major feature of the technique, and provides a new strategy by which to measure other ultraweak forces beyond the thermal noise limit.

DOI

Dimensional measurement of microform with high aspect ratio using an optically controlled particle with standing wave scale sensing

Yasuhiro Takaya, Masaki Michihata, Terutake Hayashi, Taisuke Washitani

A new measurement technique based on the localized optical interference scale formed by a standing wave is proposed. The scale is sensed using a microprobe controlled three dimensionally by radiation pressure. The feasibility of this novel probing technique is examined by measuring the depth of a micro-groove using the displacement sensing method based on a coordinate measurement system. Since the effective length of the standing wave scale is more than 250 μm, the microprobe enables us to measure a micro-fine figure with a high aspect ratio. 

DOI

Process investigations of optical trap assisted direct-write microsphere near-field nanostructuring

Karl-Heinz Leitz, Ulf Quentin, Ilya Alexeev, Michael Schmidt

In a wide range of technological and biomedical applications nanostructured surfaces become increasingly important. Laser based techniques utilizing near-field focusing of pulsed laser radiation by microsphere particles allow a robust, low-cost nanopatterning at sub-diffraction-limited resolution. In combination with optical tweezers a direct-write nanostructuring becomes feasible. In this contribution, a fundamental simulational study of optical trap assisted ultrafast direct-write near-field nanostructuring showing the influence of the main process parameters is presented.

DOI

The biomechanics of drug-treated leukemia cells investigated using optical tweezers

ZHUO L. ZHOU, BIN TANG, ALFONSO H. W. NGAN

Leukemia is a very common cancer worldwide, and different drugs have been applied to treat the disease. However, the influence of the drugs on the biomechanical properties of leukemia cells, which are related to the risk of leukostasis, is still unknown. Moreover, accurate measurement of biomechanical properties of leukemia cells is still a challenging task because of their non-adherent nature and high sensitivity to the surrounding physiological conditions. In this study, a protocol to measure the biomechanical properties of leukemia cells by performing indentation tests using optical tweezers is proposed. The biomechanical properties of normal leukemia cells and cells treated with various cancer drugs, including phorbol 12-myristate 13-acetate (PMA), all-trans retinoic acid (ATRa), Cytoxan (CTX), and Dexamethasone (DEX), were measured. The adhesion between the cells and certain proteins existing in the extracellular matrix, i.e., fibronetin and collagen I, was also characterized with the help of a static adhesion assay. It was found that after treatment by ATRa, CTX, and DEX, the cells became softer, and the adhesion between the cells and the proteins became weaker. PMA treatment caused no change in the stiffness of the HL60 cells, but increased the stiffness of the K562 cells, and increased the cell–protein adhesion of both K562 cells and HL60 cells.

DOI

Micro-Raman Spectroscopy of Silver Nanoparticle Induced Stress on Optically-Trapped Stem Cells

Aseefhali Bankapur, R. Sagar Krishnamurthy, Elsa Zachariah, Chidangil Santhosh, Basavaraj Chougule,Bhavishna Praveen, Manna Valiathan, Deepak Mathur

We report here results of a single-cell Raman spectroscopy study of stress effects induced by silver nanoparticles in human mesenchymal stem cells (hMSCs). A high-sensitivity, high-resolution Raman Tweezers set-up has been used to monitor nanoparticle-induced biochemical changes in optically-trapped single cells. Our micro-Raman spectroscopic study reveals that hMSCs treated with silver nanoparticles undergo oxidative stress at doping levels in excess of 2 µg/ml, with results of a statistical analysis of Raman spectra suggesting that the induced stress becomes more dominant at nanoparticle concentration levels above 3 µg/ml.

DOI

A hyphenated optical trap capillary electrophoresis laser induced native fluorescence system for single-cell chemical analysis

Christine Cecala , Stanislav S. Rubakhin , Jennifer M. Mitchell , Martha U. Gillette and Jonathan Sweedler

Single-cell measurements allow a unique glimpse into cell to cell heterogeneity; even small changes in selected cells can have a profound impact on an organism’s physiology. Here an integrated approach to single-cell sampling and assay are described. Capillary electrophoresis (CE) with laser-induced native fluorescence (LINF) has the sensitivity to characterize natively-fluorescent indoles and catechols within individual cells. While the separation and detection approaches are well established, the sampling and injection of individually selected cells requires new approaches. We describe an optimized system that interfaces a single-beam optical trap with CE and multichannel LINF detection. A cell is localized within the trap and then the capillary inlet is positioned near the cell using a computer-controlled micromanipulator. Hydrodynamic injection allows cell lysis to occur within the capillary inlet, followed by the CE separation and LINF detection. The use of multiple emission wavelengths allows improved analyte identification based on differences in analyte fluorescence emission profiles and migration time. The system enables injections of individual rat pinealocytes and quantification of their endogenous indoles, including serotonin, N-acetyl-serotonin, 5-hydooxyindole-3-acetic acid, tryptophol and others. The amounts detected in individual cells incubated in 5-hydroxytryptophan ranged from 10-14 mol to 10-16 mol, which is an order of magnitude lower than observed in untreated pinealocytes.

DOI

Optical trapping of carbon nanotubes and graphene

S. Vasi, M. A. Monaca, M. G. Donato, F. Bonaccorso, G. Privitera, O. Trushkevych, G. Calogero, B. Fazio, A. Irrera, M. A. Iatì, R. Saija, P. Denti, F. Borghese, P. H. Jones, A. C. Ferrari, P. G. Gucciardi, O. M. Maragò

We study optical trapping of nanotubes and graphene. We extract the distribution of both centre-of-mass and angular fluctuations from three-dimensional tracking of these optically trapped carbon nanostructures. The optical force and torque constants are measured from auto and cross-correlation of the tracking signals. We demonstrate that nanotubes enable nanometer spatial, and femto-Newton force resolution in photonic force microscopy by accurately measuring the radiation pressure in a double frequency optical tweezers. Finally, we integrate optical trapping with Raman and photoluminescence spectroscopy demonstrating the use of a Raman and photoluminescence tweezers by investigating the spectroscopy of nanotubes and graphene flakes in solution. Experimental results are compared with calculations based on electromagnetic scattering theory.

DOI

Local electric field measurements by optical tweezers

G. Pesce, B. Mandracchia, E. Orabona, G. Rusciano, L. De Stefano, A. Sasso

We report a new technique to measure direction and amplitude of electric fields generated by microelectrodes embedded in polar liquid environment, as often used in microfluidic devices. The method is based on optical tweezers which act as sensitive force transducer while a trapped charged microsphere behaves as a probe. When an electric field is applied the particles moves from its equilibrium position and finishes in a new equilibrium position where electric and optical forces are balanced. A trapped bead is moved to explore the electric field in a wide region around the microelectrodes. In such way maps of electric fields with high spatial resolution can be reconstructed even for complex electrode geometries where numerical simulation approaches can fail. Experimental results are compared with calculations based on finite element analysis simulation.

DOI

Tuning the structural and optical properties of gold/silver nanoalloys prepared by laser ablation in liquids for ultra-sensitive spectroscopy and optical trapping

E. Messina, L. D'Urso, C. Satriano, E. Fazio, M. G. Donato, B. Fazio, C. D'Andrea, O. M. Maragò, P. G. Gucciardi, G. Compagnini, F. Neri

The plasmon resonance of metallic Au/Ag alloys in the colloidal state was tuned from 400 nm to 500 nm using a laser irradiated technique, performed directly in the liquid state. Interesting optical nonlinearities, trapping effects and spectroscopic enhancements were detected as function of gold concentration in the nanoalloys. In particular a reduction of the limiting threshold was observed by increasing the gold amount. The SERS activity of the Au/Ag alloys was tested in liquid and in solid state in presence of linear carbon chains as probe molecules. The dependence of the increased Raman signals on the nanoparticle Au/Ag atomic ratio is presented and discussed. Finally preliminary studies and prospects for optical and Raman tweezers experiments are discussed.

DOI

Not just energy, but momentum and angular momentum too: mechanical effects in scattering

T. A. Nieminen, A. B. Stilgoe, N. R. Heckenberg, H. Rubinsztein-Dunlop

We review the transport and transfer of momentum and angular momentum by electromagnetic waves, and applications of the mechanical effects of scattering.

DOI

Optical trapping and optical binding using cylindrical vector beams

S. E. Skelton, M. Sergides, R. Patel, E. Karczewska, O. M. Maragò, P. H. Jones

We report on the use of cylindrical vector beams for optical manipulation of micron and sub-micron sized particles using the methods of a single-beam gradient force trap (optical tweezers) and an evanescent-field surface trap (optical binding). We have demonstrated a stable interferometric method for the synthesis of cylindrical vector beams (CVBs), and present measurements demonstrating polarization-controlled focal volume shaping using CVBs in an optical tweezers. Furthermore we show how appropriate combinations of CVBs corresponding to superpositions of optical fibre modes can be used for controlled trapping and trafficking of nanoparticles along a sub-micron diameter tapered optical fibre.

DOI

Fluorescent protein-based technologies: shedding new light on the plant endomembrane system

Imogen Sparkes, Federica Brandizzi

Without doubt, GFP and spectral derivatives have revolutionized the way biologists approach their journey toward the discovery of how plant cells function. It is fascinating that in its early days GFP was used merely for localization studies, but as time progressed researchers successfully explored new avenues to push the power of GFP technology to reach new and exciting research frontiers. This has had a profound impact on the way we can now study complex and dynamic systems such as plant endomembranes. Here we briefly describe some of the approaches where GFP has revolutionized in vivo studies of protein distribution and dynamics and focus on two emerging approaches for the application of GFP technology in plant endomembranes, namely optical tweezers and forward genetics approaches, which are based either on the light or on genetic manipulation of secretory organelles to gain insights on the factors that control their activities and integrity.

DOI

Non-specific binding of Na+ and Mg2+ to RNA determined by force spectroscopy methods

C. V. Bizarro, A. Alemany and F. Ritort

RNA duplex stability depends strongly on ionic conditions, and inside cells RNAs are exposed to both monovalent and multivalent ions. Despite recent advances, we do not have general methods to quantitatively account for the effects of monovalent and multivalent ions on RNA stability, and the thermodynamic parameters for secondary structure prediction have only been derived at 1M [Na+]. Here, by mechanically unfolding and folding a 20 bp RNA hairpin using optical tweezers, we study the RNA thermodynamics and kinetics at different monovalent and mixed monovalent/Mg2+ salt conditions. We measure the unfolding and folding rupture forces and apply Kramers theory to extract accurate information about the hairpin free energy landscape under tension at a wide range of ionic conditions. We obtain non-specific corrections for the free energy of formation of the RNA hairpin and measure how the distance of the transition state to the folded state changes with force and ionic strength. We experimentally validate the Tightly Bound Ion model and obtain values for the persistence length of ssRNA. Finally, we test the approximate rule by which the non-specific binding affinity of divalent cations at a given concentration is equivalent to that of monovalent cations taken at 100-fold concentration for small molecular constructs.

DOI

Direct Observation of Strand Passage by DNA-Topoisomerase and Its Limited Processivity

Katsunori Yogo, Taisaku Ogawa, Masahito Hayashi,Yoshie Harada, Takayuki Nishizaka, Kazuhiko Kinosita Jr

Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIα, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.51±0.33 µm (11±6 turns) of a braid was unlinked in a burst of reactions taking 8±4 s, the unlinked length being essentially independent of the enzyme concentration between 0.25–37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being ~100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of ~10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break ~10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments.

DOI

Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

Matthew H. Larson, Jing Zhou, Craig D. Kaplan, Murali Palangat, Roger D. Kornberg, Robert Landick, and Steven M. Block

During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre- or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate.

DOI

Significantly Improved Trapping Lifetime of Nanoparticles in an Optical Trap using Feedback Control

Arvind Balijepalli, Jason J Gorman, Satyandra K. Gupta, and Thomas W. LeBrun

We demonstrate an increase in trapping lifetime for optically trapped nanoparticles by more than an order of magnitude using feedback control with no corresponding increase in beam power. Langevin dynamics simulations were used to design the control law and this technique was then demonstrated experimentally for 100 nm gold particles and 350 nm silica particles. No particle escapes were detected with the controller on, leading to lower limits on the increase in lifetime for the 100 nm gold particles of 26 times (at constant average beam power) and 22 times for the 350 nm silica particles (with average beam power reduced by one third). The approach described here can be combined with other techniques such as counter propagating beams or higher-order optical modes to trap the smallest nanoparticles, and can be used to reduce optical heating of particles that are susceptible to photodamage, such as biological systems.

DOI

Assembling of three-dimensional crystals by optical depletion force induced by a single focused laser beam

Hai-Dong Deng, Guang-Can Li, Hai-Ying Liu, Qiao-Feng Dai, Li-Jun Wu, Sheng Lan, Achanta Venu Gopal, Vyacheslav A. Trofimov, and Tatiana M. Lysak

We proposed a method to assemble microspheres into a three-dimensional crystal by utilizing the giant nonequilibrium depletion force produced by nanoparticles. Such assembling was demonstrated in a colloid formed by suitably mixing silica microspheres and magnetic nanoparticles. The giant nonequilibrium depletion force was generated by quickly driving magnetic nanoparticles out of the focusing region of a laser light through both optical force and thermophoresis. The thermophoretic binding of silica beads is so tight that a colloidal photonic crystal can be achieved after complete evaporation of solvent. This technique could be employed for fabrication of colloidal photonic crystals and molecular sieves.

DOI

Plasmonic nanotweezers: strong influence of adhesion layer and nanostructure orientation on trapping performance

Brian J. Roxworthy and Kimani C. Toussaint

Using Au bowtie nanoantennas arrays (BNAs), we demonstrate that the performance and capability of plasmonic nanotweezers is strongly influenced by both the material comprising the thin adhesion layer used to fix Au to a glass substrate and the nanostructure orientation with respect to incident illumination. We find that a Ti adhesion layer provides up to 30% larger trap stiffness and efficiency compared to a Cr layer of equal thickness. Orientation causes the BNAs to operate as either (1) a 2D optical trap capable of efficient trapping and manipulation of particles as small as 300 nm in diameter, or (2) a quasi-3D trap, with the additional capacity for size-dependent particle sorting utilizing axial Rayleigh-Bénard convection currents caused by heat generation. We show that heat generation is not necessarily deleterious to plasmonic nanotweezers and achieve dexterous manipulation of nanoparticles with non-resonant illumination of BNAs.

DOI

Optical trapping force reduction and manipulation of nanoporous beads

Tao Wang, Fan Jiang, Stefan Oehrlein, Erliang Zeng, Ryan Kershner, and Franco Cerrina

We studied the interaction of infrared optical traps with controlled-pore glass (CPG) beads in aqueous medium. The lateral optical trapping force and stiffness were experimentally found considerably smaller than those of their solid counterparts. The simulation using an average refractive index revealed significant losses of effective trapping efficiency, which quantitatively agreed well with experimentally fitted curves. This effect was ascribed to the reduced relative refractive index of medium-filled CPG beads with respect to the medium. Combining optical trapping with mechanical confinements, we demonstrated a microfluidic platform allowing for the synthesis of multiple DNA oligonucleotide sequences on individual beads of interest.

DOI

Optical delivery of nanospheres using arbitrary bending nanofibers

Ying Li, Linlin Xu and Baojun Li

This work reports an optical delivery of about 700-nm diameter polystyrene spheres along arbitrary bending nanofibers (600 nm in diameter) including complete loop structure. Dependence of bending loss on bending radii and central angles of the nanofiber has also been investigated. The results show that, for a specific input optical power, there is a corresponding minimum bending radius for optical trapping and delivery. In other words, for a specific input optical power, when the bending radius of the nanofiber is larger than the minimum bending radius, the 700-nm diameter nanospheres can be trapped and delivered along the bending nanofiber. Vice versa, the nanospheres will be escaped from the bending nanofiber during the delivery process because of relatively large bending loss.

DOI

Parallel optical trap assisted nanopatterning on rough surfaces

Y-C Tsai, K-H Leitz, R Fardel, A Otto, M Schmidt and C B Arnold

There exist many optical lithography techniques for generating nanostructures on hard, flat surfaces over large areas. However, few techniques are able to create such patterns on soft materials or surfaces with pre-existing structure. To address this need, we demonstrate the use of parallel optical trap assisted nanopatterning (OTAN) to provide an efficient and robust direct-write method of producing nanoscale features without the need for focal plane adjustment. Parallel patterning on model surfaces of polyimide with vertical steps greater than 1.5 µm shows a feature size uncertainty better than 4% across the step and lateral positional accuracy of 25 nm. A Brownian motion model is used to describe the positional accuracy enabling one to predict how variation in system parameters will affect the nanopatterning results. These combined results suggest that OTAN is a viable technique for massively parallel direct-write nanolithography on non-traditional surfaces.

DOI

Improvement of optical trapping effect by using the focused high-order Laguerre–Gaussian beams

Hai-Shui Chai, Li-Gang Wang

We investigate the optical trapping effect of high-order Laguerre–Gaussian (LG) beams acting on a dielectric sphere in Rayleigh regime. For LG beams with the azimuthal mode index l = 0, it is found that under the same input power, the transverse trapping effect can be enhanced several times with increasing the radial mode index p, compared with that of the Gaussian beam; while its axial trapping effect is exactly the same as that of Gaussian beam, although the central trapping region reduces as p increases. For LG beams with l ≥ 1, we find that the maximal transverse gradient forces increase with the increasing of p and the axial radiation forces reduces slightly, therefore an optimal choose on p and l is necessary for obtaining an optimal optical guiding. Our result is useful for analyzing the trapping efficiency of LG beams applied in micromanipulation technologies.

DOI

The self-orientation of mammalian cells in optical tweezers—the importance of the nucleus

Nicolas M B Perney, Peter Horak, Neil A Hanley and Tracy Melvin

Here we present the first evidence showing that eukaryotic cells can be stably trapped in a single focused Gaussian beam with an orientation that is defined by the nucleus. A mammalian eukaryotic cell (in suspension) is trapped and is re-oriented in the focus of a linearly polarized Gaussian beam with a waist of dimension smaller than the radius of the nucleus. The cell reaches a position relative to the focus that is dictated by the nucleus and nuclear components. Our studies illustrate that the force exerted by the optical tweezers at locations within the cell can be predicted theoretically; the data obtained in this way is consistent with the experimental observations.

DOI

Parallel Raman microspectroscopy using programmable multipoint illumination

Ji Qi and Wei-Chuan Shih

We present a novel parallel Raman microspectroscopy scheme for simultaneously collecting Raman spectra from multiple points. This scheme is realized by projectinga multiple-point laser illumination pattern using a spatial light modulator (SLM) and wide-field Raman imaging collection. We demonstrate the performance of this scheme using uniform samples, trapped polymer microparticles and fixed polymer microparticles with mixed molecular composition within a 50×50  μm2 field of view. This scheme enables the acquisition of Raman spectra from as many as 40 points simultaneously using a single illumination pattern and detector recording frame without scanning.

DOI

DNA-Cisplatin interaction studied with single molecule stretching experiments

Fabiano Crisafuli, Estevan Cesconetto, Esio Ramos and Marcio Santos Rocha

By performing single molecule stretching experiments with optical tweezers, we have studied the changes on the mechanical properties of DNA-cisplatin complexes as a function of some variables of interest such as the drug diffusion time and concentration in the sample. We propose a model to explain the behavior of the persistence length as a function of the drug concentration, extracting the binding data from pure mechanical measurements. Such analysis has allowed us to show that cisplatin binds cooperatively to the DNA molecule. In addition, DNA compactation by the action of the drug was recovered here for our experimental conditions by studying the kinetics of some mechanical properties such as the radius of gyration and the end-to-end distance.

DOI

Single-molecule observation of helix staggering, sliding, and coiled coil misfolding

Zhiqun Xi, Ying Gao, George Sirinakis, Honglian Guo, and Yongli Zhang

The biological functions of coiled coils generally depend on efficient folding and perfect pairing of their α-helices. Dynamic changes in the helical registry that lead to staggered helices have only been proposed for a few special systems and not found in generic coiled coils. Here, we report our observations of multiple staggered helical structures of two canonical coiled coils. The partially folded structures are formed predominantly by coiled coil misfolding and occasionally by helix sliding. Using high-resolution optical tweezers, we characterized their energies and transition kinetics at a single-molecule level. The staggered states occur less than 2% of the time and about 0.1% of the time at zero force. We conclude that dynamic changes in helical registry may be a general property of coiled coils. Our findings should have broad and unique implications in functions and dysfunctions of proteins containing coiled coils.

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Bidirectional Optical Sorting of Gold Nanoparticles

M. Ploschner, T. Čižmár, M. Mazilu, A. Di Falco, and K. Dholakia

We present a generic technique allowing size-based all-optical sorting of gold nanoparticles. Optical forces acting on metallic nanoparticles are substantially enhanced when they are illuminated at a wavelength near the plasmon resonance, as determined by the particle’s geometry. Exploiting these resonances, we realize sorting in a system of two counter-propagating evanescent waves, each at different wavelengths that selectively guide nanoparticles of different sizes in opposite directions. We validate this concept by demonstrating bidirectional sorting of gold nanoparticles of either 150 or 130 nm in diameter from those of 100 nm in diameter within a mixture.

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Efficient and low cost multiple optical trap, based on interference

D.G. Kotsifaki, M. Makropoulou, A.A. Serafetinides

Interference fringes provide the means for simultaneous, multiple–micromanipulation, of particles. In biomedical investigations, multiple interference optical trapping gives the feasibility to trap a large number of biological substances. In this paper we describe a simple method for creating multiple optical tweezers from a single laser beam, using a system of ten transparent glass beam splitters. The experimental set up was tested, on polystyrene beads of intermediate size. We also measured the optical forces exerted by the fringes, in positions where the three and two dimensions optical trap was stable, and we evaluated the experimentally obtained optical efficiency. We notice that the experimentally optical efficiency value for the higher order fringes decreases.

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Kinesin backsteps

Nicholas J. Carter and Robert A. Cross

Kinesin-1 is a walking machine that takes ~8 nm steps along microtubules. Some aspects of the molecular mechanism of walking are now clear, but many are not. In the present paper, we discuss currently controversial points, focusing on the pathways by which kinesin takes occasional backsteps. Backsteps represent failures of the forwards-biasing mechanism. By studying the mechanochemistry of backstepping, one can learn much about the underlying molecular mechanisms responsible for forwards directional bias in the walking action.

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Dynein Tethers and Stabilizes Dynamic Microtubule Plus Ends

Adam G. Hendricks, Jacob E. Lazarus, Eran Perlson, Melissa K. Gardner, David J. Odde, Yale E. Goldman, Erika L.F. Holzbaur

Microtubules undergo alternating periods of growth and shortening, known as dynamic instability. These dynamics allow microtubule plus ends to explore cellular space. The “search and capture” model posits that selective anchoring of microtubule plus ends at the cell cortex may contribute to cell polarization, spindle orientation, or targeted trafficking to specific cellular domains. Whereas cytoplasmic dynein is primarily known as a minus-end-directed microtubule motor for organelle transport, cortically localized dynein has been shown to capture and tether microtubules at the cell periphery in both dividing and interphase cells. To explore the mechanism involved, we developed a minimal in vitro system, with dynein-bound beads positioned near microtubule plus ends using an optical trap. Dynein induced a significant reduction in the lateral diffusion of microtubule ends, distinct from the effects of other microtubule-associated proteins such as kinesin-1 and EB1. In assays with dynamic microtubules, dynein delayed barrier-induced catastrophe of microtubules. This effect was ATP dependent, indicating that dynein motor activity was required. Computational modeling suggests that dynein delays catastrophe by exerting tension on individual protofilaments, leading to microtubule stabilization. Thus, dynein-mediated capture and tethering of microtubules at the cortex can lead to enhanced stability of dynamic plus ends.

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The mechanical properties of kinesin-1: a holistic approach

George M. Jeppesen and J.K. Heinrich Hoerber

During the last 25 years, a vast amount of research has gone into understanding the mechanochemical cycle of kinesin-1 and similar processive motor proteins. An experimental method that has been widely used to this effect is the in vitro study of kinesin-1 molecules moving along microtubules while pulling a bead, the position of which is monitored optically while trapped in a laser focus. Analysing results from such experiments, in which thermally excited water molecules are violently buffeting the system components, can be quite difficult. At low loads, the effect of the mechanical properties of the entire molecule must be taken into account, as stalk compliance means the bead position recorded is only weakly coupled to the movement of the motor domains, the sites of ATP hydrolysis and microtubule binding. In the present review, findings on the mechanical and functional properties of the various domains of full-length kinesin-1 molecules are summarized and a computer model is presented that uses this information to simulate the motion of a bead carried by a kinesin molecule along a microtubule, with and without a weak optical trap present. A video sequence made from individual steps of the simulation gives a three-dimensional visual insight into these types of experiment at the molecular level.

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Negative radiation pressure and negative effective refractive index via dielectric birefringence

Jonathan Nemirovsky, Mikael C. Rechtsman, and Mordechai Segev

We show that light guided in a planar dielectric slab geometry incorporating a biaxial medium has lossless modes with group and phase velocities in opposite directions. Particles in a vacuum gap inserted into the structure experience negative radiation pressure: the particles are pulled by light rather than pushed by it. This effectively one-dimensional dielectric structure represents a new geometry for achieving negative radiation pressure in a wide range of frequencies with minimal loss. Moreover, this geometry provides a straightforward platform for experimentally resolving the Abrahams-Minkowski dilemma.

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Expanding the toolbox for nanoparticle trapping and spectroscopy with holographic optical tweezers

M Dienerowitz, G Gibson, F Dienerowitz and M Padgett

We have developed a workstation based on holographic tweezers to optically trap, move and characterize metal nanoparticles. Our advanced darkfield imaging system allows us to simultaneously image and take spectra of single trapped metal nanoparticles. We take advantage of the beamshaping abilities of the spatial light modulator and correct for aberrations of the trapping optics. We monitor the improvement of the optical trap with video-based nanoparticle tracking. Furthermore we theoretically assess the capabilities and limitations of video-based tracking for nanoparticle position detection, in particular with respect to acquisition frequencies below the corner frequency.

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Single cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB)

Sunje J. Pamp, Eoghan D. Harrington, Stephen R. Quake, David A. Relman and Paul C. Blainey

Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated CandidatusArthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which "holdfasts" and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences to each other, as well as to recently-published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell-cycle control, and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a predicted myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract.

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Generalized Mie theory of optical forces

Alessandro Salandrino, Shima Fardad, and Demetrios N. Christodoulides

The theory of optical forces on spherical scatterers is here generalized to arbitrary incident fields. The interaction between spherical harmonics of different order, and the degree and azimuthal parity, is studied in detail. The resulting force from all the contributing components is presented in analytical form. A further generalization of this formulation to nonspherical scatterers is also discussed.

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Probing the dynamics of an optically trapped particle by phase sensitive back focal plane interferometry

Basudev Roy, Sambit Bikas Pal, Arijit Haldar, Ratnesh Kumar Gupta, Nirmalya Ghosh, and Ayan Banerjee
The dynamics of an optically trapped particle are often determined by measuring intensity shifts of the back-scattered light from the particle using position sensitive detectors. We present a technique which measures the phase of the back-scattered light using balanced detection in an external Mach-Zehnder interferometer scheme where we separate out and beat the scattered light from the particle and that from the top surface of our trapping chamber. The technique has improved axial motion resolution over intensity-based detection, and can also be used to measure lateral motion of the trapped particle. In addition, we are able to track the Brownian motion of trapped 1.1 and 3 μm diameter particles from the phase jitter and show that, similar to intensity-based measurements, phase measurements can also be used to simultaneously determine displacements of the trapped particle as well as the spring constant of the trap. For lateral displacements, we have matched our experimental results with a simulation of the overall phase contour of the back-scattered light by using plane wave decomposition in conjunction with Mie scattering theory. The position resolution is limited by path drifts of the interferometer which we have presently reduced to demonstrate the capability of sub-nm displacement resolution in the axial direction for 1.1 μm diameter particles by locking the interferometer to a frequency stabilized diode laser.

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Mechanochemistry of single red blood cells monitored using Raman tweezers

Saurabh Raj, Mónica Marro, Michal Wojdyla, and Dmitri Petrov

Two microparticles were biochemically attached to a red blood cell at diametrically opposite parts and held by optical traps allowing to impose deformations. The cell deformation was monitored from the microscopy images. Raman spectra of the cell under tunable deformations were studied. Vibrational spectra analysis at different stretching states was supported with two statistical methods. Principal Component Analysis distinguishes the most prominent changes in spectra while 2D correlation technique monitors the evolution of Raman bands during stretching. The measurements show significant changes in the cell chemical structure with stretching however the changes saturate above 20% of cell deformation. Mechanical deformation of the cell mainly affects the bands corresponding to hemoglobin but contributions from spectrin and membrane proteins can not be excluded. The saturation of bands at higher deformations suggests some structural relaxation that RBC has to undergo to bear extra load. The results confirm widely accepted belief that spectrin released from membrane proteins allows for significant shape changes of the cells. We therefore tentatively suggest that interaction between membrane and cytoskeleton during deformation can be efficiently probed by confocal Raman spectroscopy, in particular via the peak around 1035 cm−1.

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Force–Fluctuation Relation of a Single DNA Molecule

Takeshi Baba, Takahiro Sakaue, and Yoshihiro Murayama

We observed transverse fluctuations of single DNA molecules by fluorescence microscopy. The end-to-end distance of DNA molecules was varied by using dual trap optical tweezers, and the force–fluctuation relation was experimentally obtained in wide ranges of the force regime. In strong force regime, the theory of a stretched worm-like chain with fixed both ends explains the experimental results. On the other hand, in the low force regime, the fluctuations approach the value for an ideal ring polymer. We introduce an interpolate formula for the force–fluctuation relation by considering strong and low force limits, which captures the overall trend of experimental results. The proposed force–fluctuation relation will be useful for quantitative discussions in various sectors of polymer physics and biological processes which involve the conformation change of DNA and other biopolymers, where the loading and the fluctuation are relevant factors.

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Selection and Characterization of Aerosol Particle Size Using a Bessel Beam Optical Trap for Single Particle Analysis

Antonia E. Carruthers, Jim S. Walker, Abby Casey, Andrew Orr-Ewing and Jonathan P Reid

Bessel beams were used to create a counter-propagating optical trap for capturing and manipulating aerosol particles. Aerosol droplets were characterized through measurement of the elastic scattered light at three wavelengths; the trapping wavelength of 532 nm was used in conjunction with two probe beams at 405 nm and 633 nm to reduce the uncertainty in estimating droplet radii of 1 μm or less. Control of the aerosol size distribution sampled by the counter-propagating trap was demonstrated by varying the trapping beam core diameters and intensities. Smaller droplet sizes were preferentially selected with a 1.7 μm core diameter compared to cores of 2.7 μm and 4.5 μm. Further, an increase in core intensity was shown to broaden the range in droplet sizes that were optically trapped. The possibility of using such an approach to isolate and analyze the properties of single accumulation mode aerosol particles is discussed.

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Unconventional structure-assisted optical manipulation of high-index nanowires in liquid crystals

David Engström, Michael C.M. Varney, Martin Persson, Rahul P. Trivedi, Kris A. Bertness, Mattias Goksör, and Ivan I. Smalyukh

Stable optical trapping and manipulation of high-index particles in low-index host media is often impossible due to the dominance of scattering forces over gradient forces. Here we explore optical manipulation in liquid crystalline structured hosts and show that robust optical manipulation of high-index particles, such as GaN nanowires, is enabled by laser-induced distortions in long-range molecular alignment, via coupling of translational and rotational motions due to helicoidal molecular arrangement, or due to elastic repulsive interactions with confining substrates. Anisotropy of the viscoelastic liquid crystal medium and particle shape give rise to a number of robust unconventional trapping capabilities, which we use to characterize defect structures and study rheological properties of various thermotropic liquid crystals.

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Videomicroscopy calibration of optical tweezers by position autocorrelation function analysis

P. S. Alves and M. S. Rocha

We present a simple method to calibrate optical tweezers by using only videomicroscopy to measure the position autocorrelation function of the trapped bead in the potential well of the tweezers. To accomplish this task, we use a high-speed camera with ∼500 fps (frames per second), which provides a precise measurement of the relaxation time of the bead Brownian fluctuations. We also study the variation of the trap stiffness as a function of some parameters of interest such as the laser power and the distance from the bead center to the microscope coverslip, showing that the presented method returns precise results.

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Self-assembly of microparticles in stable ring structures in an optical trap

Arijit Haldar, Sambit Bikas Pal, Basudev Roy, S. Dutta Gupta, and Ayan Banerjee

Microparticle self assembly under the influence of optical forces produced by higher-order optical beams or by projection of a hologram into the trapping volume is well known. In this paper, we report the spontaneous formation of a ring of identical microspheres (each with diameter 1.1 μm) in conventional single-beam optical tweezers with a usual TEM00Gaussian beam coupled into a sample chamber having a standing wave geometry with a cover slip and glass slide. The effects of different experimental parameters on the ring formation are studied extensively. The experimental observations are backed by theoretical simulations based on a plane wave decomposition of the forward- and backward-propagating Gaussian beams. The ring patterns are shown to be caused due to geometrical aberrations produced by focusing the Gaussian beam using a high-numerical-aperture microscope objective into stratified media. It is found that the thickness of the stratified media and the standing wave geometry itself play a critical role in the formation of stable ring structures. These structures could be used in the study of optical binding, as well as of biological interactions between cells in an optical trap.

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Absolute calibration of optical tweezers including aberrations

R. S. Dutra, N. B. Viana, P. A. Maia Neto, and H. M. Nussenzveig

We extend a previous proposal for absolute calibration of optical tweezers by including optical setup aberrations into the first-principles theory, with no fitting parameters. Astigmatism, the dominant term, is determined from images of the focused laser spot. Correcting it can substantially increase stiffness. Comparison with experimental results yields agreement within error bars for a broad range of bead sizes and trap heights, as well as different polarizations. Absolute calibration is established as a reliable and practical method for applications and design of optical tweezers systems.

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Optical force lateral push–pulling using focus positioning

Murat Muradoglu, Wesley Sung-Yee Chiu, and Tuck Wah Ng

The placement of the beam focus corresponding with the center of living matter (such as cells) in an optical tweezer can result in photodamage. We advance a scheme here that locates the focus of the beam either above or below the matter to pull and push relative to the beam axis in a predominant lateral sense based on the resultant action of scattering and gradient forces. Switching to a laser that acts oppositely serves to restore the axial position of the matter. Although an exact value could not be derived due to the statistical nature of Brownian perturbations and time frame considerations, we simulated the optical force fields to visualize the effective force envelope. The lateral optical push–pull operation was conducted experimentally on polystyrene beads in which the motion manipulation efficacy was characterized.

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