Tuesday, April 30, 2013

Design of hybrid optical tweezers system for controlled three-dimensional micromanipulation

Yoshio Tanaka; Shogo Tsutsui; Hiroyuki Kitajima
Three-dimensional (3D) micro/nano-manipulation using optical tweezers is a significant technique for various scientific fields ranging from biology to nanotechnology. For the dynamic handling of multiple/individual micro-objects in a true 3D working space, we present an improved hybrid optical tweezers system consisting of two multibeam techniques. These two techniques include the generalized phase contrast method with a spatial light modulator and the time-shared scanning method with a two-axis steering mirror and an electrically focus-tunable lens. Unlike our previously reported system that could only handle micro-objects in a two and half dimensional working space, the present system has high versatility for controlled manipulation of multiple micro-objects in a true 3D working space. The controlled rotation of five beads forming a pentagon, that of four beads forming a tetrahedron about arbitrary axes, and the fully automated assembly and subsequent 3D translation of micro-bead arrays are successfully demonstrated as part of the 3D manipulation experiment.


Stochastic Hydrodynamic Synchronization in Rotating Energy Landscapes

N. Koumakis and R. Di Leonardo

Hydrodynamic synchronization provides a general mechanism for the spontaneous emergence of coherent beating states in independently driven mesoscopic oscillators. A complete physical picture of those phenomena is of definite importance to the understanding of biological cooperative motions of cilia and flagella. Moreover, it can potentially suggest novel routes to exploit synchronization in technological applications of soft matter. We demonstrate that driving colloidal particles in rotating energy landscapes results in a strong tendency towards synchronization, favoring states where all beads rotate in phase. The resulting dynamics can be described in terms of activated jumps with transition rates that are strongly affected by hydrodynamics leading to an increased probability and lifetime of the synchronous states. Using holographic optical tweezers we quantitatively verify our predictions in a variety of spatial configurations of rotors.


Chirality Screening and Metastable States in Chiral Nematic Colloids

V. S. R. Jampani, M. Škarabot, S. Čopar, S. Žumer, and I. Muševič 
Hydrodynamic synchronization provides a general mechanism for the spontaneous emergence of coherent beating states in independently driven mesoscopic oscillators. A complete physical picture of those phenomena is of definite importance to the understanding of biological cooperative motions of cilia and flagella. Moreover, it can potentially suggest novel routes to exploit synchronization in technological applications of soft matter. We demonstrate that driving colloidal particles in rotating energy landscapes results in a strong tendency towards synchronization, favoring states where all beads rotate in phase. The resulting dynamics can be described in terms of activated jumps with transition rates that are strongly affected by hydrodynamics leading to an increased probability and lifetime of the synchronous states. Using holographic optical tweezers we quantitatively verify our predictions in a variety of spatial configurations of rotors.

Mapping AC Electroosmotic Flow at the Dielectrophoresis Crossover Frequency of a Colloidal Probe

Jingyu Wang, Ming-Tzo Wei, Joel A. Cohen, H. Daniel Ou-Yang
AC electroosmotic (ACEO) flow above the gap between coplanar electrodes is mapped by the measurement of Stokes forces on an optically-trapped polystyrene colloidal particle. E2-dependent forces on the probe particle are selected by amplitude modulation (AM) of the ACEO electric field (E) and lock-in detection at twice the AM frequency. E2-dependent dielectrophoresis (DEP) of the probe is eliminated by driving the ACEO at the probe's DEP crossover frequency. The location-independent DEP crossover frequency is determined, in a separate experiment, as the limiting frequency of zero horizontal force as the probe is moved toward the midpoint between the electrodes. The ACEO velocity field, uncoupled from probe DEP effects, was mapped in the region 1–9 μm above a 28 μm gap between the electrodes. By use of variously-sized probes, each at its DEP crossover frequency, the frequency dependence of the ACEO flow was determined at a point 3 μm above the electrode gap and 4 μm from an electrode tip. At this location the ACEO flow was maximal at ∼117 kHz for a low-salt solution. This optical trapping method, by eliminating DEP forces on the probe, provides unambiguous mapping of the ACEO velocity field.

Thermorheology of living cells—impact of temperature variations on cell mechanics

Tobias R Kießling, Roland Stange, Josef A Käs and Anatol W Fritsch
Upon temperature changes, we observe a systematic shift of creep compliance curves J(t) for single living breast epithelial cells. We use a dual-beam laser trap (optical stretcher) to induce temperature jumps within milliseconds, while simultaneously measuring the mechanical response of whole cells to optical force. The cellular mechanical response was found to differ between sudden temperature changes compared to slow, long-term changes implying adaptation of cytoskeletal structure. Interpreting optically induced cell deformation as a thermorheological experiment allows us to consistently explain data on the basis of time–temperature superposition, well known from classical polymer physics. Measured time shift factors give access to the activation energy of the viscous flow of MCF-10A breast cells, which was determined to be ≈80 kJ mol−1. The presented measurements highlight the fundamental role that temperature plays for the deformability of cellular matter. We propose thermorheology as a powerful concept to assess the inherent material properties of living cells and to investigate cell regulatory responses upon environmental changes.

Giant resonant light forces in microspherical photonics

Yangcheng Li, Oleksiy V Svitelskiy, Alexey V Maslov, David Carnegie, Edik Rafailov and Vasily N Astratov
Resonant light pressure effects can open new degrees of freedom in optical manipulation with microparticles, but they have been traditionally considered as relatively subtle effects. Using a simplified two-dimensional model of surface electromagnetic waves evanescently coupled to whispering gallery modes (WGMs) in transparent circular cavities, we show that under resonant conditions the peaks of the optical forces can approach theoretical limits imposed by the momentum conservation law on totally absorbing particles. Experimentally, we proved the existence of strong peaks of the optical forces by studying the optical propulsion of dielectric microspheres along tapered microfibers. We observed giant optical propelling velocities ~0.45 mm s−1 for some of the 15-20 µm polystyrene microspheres in water for guided powers limited at ~43 mW. Such velocities exceed previous observations by more than an order of magnitude, thereby providing evidence for the strongly enhanced resonant optical forces. We analyzed the statistical properties of the velocity distribution function measured for slightly disordered (~1% size variations) ensembles of microspheres with mean diameters varying from 3 to 20 µm. These results demonstrate a principal possibility of optical sorting of microspheres with the positions of WGM resonances overlapped at the wavelength of the laser source. They can be used as building blocks of the lossless coupled resonator optical waveguides and various integrated optoelectronics devices.

Monday, April 29, 2013

Microfluidic cell sorter for use in developing red fluorescent proteins with improved photostability

Lloyd M Davis, Jennifer L Lubbeck, Kevin M Dean, Amy Palmer and Ralph Jimenez

This paper presents a novel microfluidic cytometer for mammalian cells that rapidly measures the irreversible photobleaching of red fluorescent proteins expressed within each cell and achieves high purity (>99%) selection of individual cells based on these measurements. The selection is achieved by using sub-millisecond timed control of a piezo-tilt mirror to steer a focused 1064-nm laser spot for optical gradient force switching following analysis of the fluorescence signals from passage of the cell through a series of 532-nm laser beams. In transit through each beam, the fluorescent proteins within the cell undergo conversion to dark states, but the microfluidic chip enables the cell to pass sufficiently slowly that recovery from reversible dark states occurs between beams, thereby enabling irreversible photobleaching to be quantified separately from the reversible dark-state conversion. The microfluidic platform achieves sorting of samples down to sub-millilitre volumes with minimal loss, wherein collected cells remain alive and can subsequently proliferate. The instrument provides a unique first tool for rapid selection of individual mammalian cells on the merits of photostability and is likely to form the basis of subsequent lab-on-a-chip platforms that combine photobleaching with other spectroscopic measurements for on-going research to develop advanced red fluorescent proteins by screening of genetic libraries.

UV and visible light induced fission of azobenzene-containing polymer vesicles

Kun Chen, Guosheng Xue, Guangyong Shen, Jun Cai, Gang Zou, Yinmei Li and Qijin Zhang
The azobenzene-containing polymer vesicles show a photo-induced fission with the irradiation of UV light or visible light. With the help of Laser-trapping Raman spectroscopy (LTRS), we find that photoisomerization of azobenzene is not the only cause of the membrane deformation.

Long tethers provide high-force coupling of the Dam1 ring to shortening microtubules

Vladimir A. Volkov, Anatoly V. Zaytsev, Nikita Gudimchuk, Paula M. Grissom, Alexander L. Gintsburg, Fazly I. Ataullakhanov, J. Richard McIntosh, and Ekaterina L. Grishchuk
Microtubule kinetochore attachments are essential for accurate mitosis, but how these force-generating connections move chromosomes remains poorly understood. Processive motion at shortening microtubule ends can be reconstituted in vitro using microbeads conjugated to the budding yeast kinetochore protein Dam1, which forms microtubule-encircling rings. Here, we report that, when Dam1 is linked to a bead cargo by elongated protein tethers, the maximum force transmitted from a disassembling microtubule increases sixfold compared with a short tether. We interpret this significant improvement with a theory that considers the geometry and mechanics of the microtubule–ring–bead system. Our results show the importance of fibrillar links in tethering microtubule ends to cargo: fibrils enable the cargo to align coaxially with the microtubule, thereby increasing the stability of attachment and the mechanical work that it can do. The force-transducing characteristics of fibril-tethered Dam1 are similar to the analogous properties of purified yeast kinetochores, suggesting that a tethered Dam1 ring comprises the main force-bearing unit of the native attachment.

Trapping red blood cells in living animals using optical tweezers

Min-Cheng Zhong, Xun-Bin Wei, Jin-Hua Zhou, Zi-Qiang Wang & Yin-Mei Li
The recent development of non-invasive imaging techniques has enabled the visualization of molecular events underlying cellular processes in live cells. Although microscopic objects can be readily manipulated at the cellular level, additional physiological insight is likely to be gained by manipulation of cells in vivo, which has not been achieved so far. Here we use infrared optical tweezers to trap and manipulate red blood cells within subdermal capillaries in living mice. We realize a non-contact micro-operation that results in the clearing of a blocked microvessel. Furthermore, we estimate the optical trap stiffness in the capillary. Our work expands the application of optical tweezers to the study of live cell dynamics in animals.

Direct measurements of colloidal hydrodynamics near flat boundaries using oscillating optical tweezers

Chungil Ha, H.D. Ou-Yang, Hyuk Kyu Pak 

We studied the hydrodynamic interaction between a colloidal particle close to flat rigid boundaries and the surrounding fluid using oscillating optical tweezers. A colloidal particle located near walls provides a model system to study the behavior of more complex systems whose boundaries can be modeled as effective walls, such as a blood tube, cell membrane, and capillary tube in bio-MEMS. In this study, we measure the hydrodynamic interaction directly without using the Stokes-Einstein relation. Two different cases are studied: a colloidal sphere near a single flat wall and a colloidal sphere located at the midplane between two flat walls. The colloidal hydrodynamics is measured as a function of the distance between the particle and the walls, and is compared with the theoretical results from well-define hydrodynamics approximations. 

Tuesday, April 23, 2013

Anomalous diffusion and power-law relaxation of the time averaged mean squared displacement in worm-like micellar solutions

Jae-Hyung Jeon, Natascha Leijnse, Lene B Oddershede and Ralf Metzler
We report the results of single tracer particle tracking by optical tweezers and video microscopy in micellar solutions. From careful analysis in terms of different stochastic models, we show that the polystyrene tracer beads of size 0.52–2.5 μm after short-time normal diffusion turn over to perform anomalous diffusion of the form 〈r2(t)〉 tα with α ≈ 0.3. This free anomalous diffusion is ergodic and consistent with a description in terms of the generalized Langevin equation with a power-law memory kernel. With optical tweezers tracking, we unveil a power-law relaxation over several decades in time to the thermal plateau value under the confinement of the harmonic tweezer potential, as predicted previously (Phys. Rev. E 85 021147 (2012)). After the subdiffusive motion in the millisecond range, the motion becomes faster and turns either back to normal Brownian diffusion or to even faster superdiffusion, depending on the size of the tracer beads.

Interrelation between various types of optically induced forces

V.P. Torchigin, A.V. Torchigin
Optically induced forces applied to a transparent optical medium are analyzed. It is shown on the basis of various approaches that the density of optically induced forces applied to a homogeneous optical medium located in an inhomogeneous electrical field is equal to zero at a steady-state. This result contradicts that obtained by means of an approach based on the Lorentz density force. An explanation is presented that the Lorentz density force is compensated at a steady-state by other kind of optically induced force. Thus, a calculation of optically induced force based on the approach using the Lorentz force is inconsistent.

Three dimensional live cell lithography

Anna Linnenberger, Martha I. Bodine, Callie Fiedler, Justine J. Roberts, Stacey C. Skaalure, Joseph P. Quinn, Stephanie J. Bryant, Michael Cole, and Robert R. McLeod

We investigate holographic optical trapping combined with step-and-repeat maskless projection stereolithography for fine control of 3D position of living cells within a 3D microstructured hydrogel. C2C12 myoblast cells were chosen as a demonstration platform since their development into multinucleated myotubes requires linear arrangements of myoblasts. C2C12 cells are positioned in the monomer solution with multiple optical traps at 1064 nm and then encapsulated by photopolymerization of monomer via projection of a 512x512 spatial light modulator illuminated at 405 nm. High 405 nm sensitivity and complete insensitivity to 1064 nm was enabled by a lithium acylphosphinate (LAP) salt photoinitiator. These wavelengths, in addition to brightfield imaging with a white light LED, could be simultaneously focused by a single oil immersion objective. Large lateral dimensions of the patterned gel/cell structure are achieved by x and y step-and-repeat process. Large thickness is achieved through multi-layer stereolithography, allowing fabrication of precisely-arranged 3D live cell scaffolds with micron-scale structure and millimeter dimensions. Cells are shown to retain viability after the trapping and encapsulation procedure.

Monday, April 22, 2013

Dynamic microbead arrays for biosensing applications

Mael Manesse, Aaron F. Phillips, Christopher N. LaFratta, Manuel A. Palacios, Ryan B. Hayman and David R Walt
In this paper we present the development of an optical tweezers platform capable of creating on-demand dynamic microbead arrays for the multiplexed detection of biomolecules. We demonstrate the use of time-shared optical tweezers to dynamically assemble arrays of sensing microspheres, while simultaneously recording fluorescence signals in real time. The detection system is able to achieve multiplexing by using quantum dot nanocrystals as both signaling probes and encoding labels on the surface of the trapped microbeads. The encoding can be further extended by using a range of bead sizes. Finally, the platform is used to detect and identify three genes expressed by pathogenic strains of Escherichia coli O157:H7. The in situ actuation enabled by the optical tweezers, combined with multiplexed fluorescence detection offers a new tool, readily adaptable to biosensing applications in microfluidic devices, and could potentially enable the development of on-demand diagnostics platforms.

The transition from liquid to solid-like behaviour in ultrahigh viscosity aerosol particles

R. M. Power, S. H. Simpson, J. P. Reid and A. J. Hudson
For the first time, a measurement of the viscosity of microparticles composed of Newtonian fluids has been made over a range of 12 orders of magnitude (10−3 to 109 Pa s), extending from dilute aqueous solutions to the solid-like behaviour expected on approaching a glass transition. Using holographic optical tweezers to induce coalescence between two aerosol particles (volume <500 femtolitres), we observe the composite particle relax to a sphere over a timescale from 10−7 to 105 s, dependent on viscosity. The damped oscillations in shape illustrate the interplay of surface capillary forces and bulk fluid flow as the relaxation progresses. Viscosity values estimated from the extrapolation of measurements from macroscopic binary aqueous solutions of sucrose are shown to diverge from the microparticle measurements by as much as five orders of magnitude in the limit of ultrahigh solute supersaturation and viscosity. This is shown to be a consequence of the sensitivity of the viscosity to the composition of the particles, specifically the water content, and the often incorrect compositional dependence on water activity that are assumed to characterise aerosols and amorphous phases under dry conditions. For ternary mixtures of sodium chloride, sucrose and water, the measured viscosities similarly diverge from model predictions by up to three orders of magnitude. The Stokes–Einstein treatment for relating the diffusivity of water in sucrose droplets to the particle viscosity is found to depart from the measured viscosities by more than one order of magnitude when the viscosity exceeds 10 Pa s and up to six orders of magnitude at the highest viscosities accessed. Coalescence is shown to proceed with unit efficiency even up to the highest accessible viscosity. These measurements provide the first comprehensive account of the change in a material property accompanying a transition from a dilute solution to an amorphous semi-solid state using aerosol particles to probe the change in rheological properties.


Optical mobility of blood cells for label-free cell separation applications

Kyung Heon Lee, Kang Soo Lee, Jin Ho Jung, Cheong Bong Chang, and Hyung Jin Sung

This paper describes the optical mobilities of blood cell components. Blood cells are heterogeneous, and their optical behaviors depend on size, morphology, and other optical properties. In a step toward the label-free separation of blood cells, the optical mobility resulting from the optical scattering and cell properties was derived and evaluated for each cell component. The optical mobilities of red blood cells, lymphocytes, granulocytes, and monocytes were measured under various flow conditions of a cross-type optical particle separator.

Permanently Fixing or Reversible Trap-and-Release of DNA Micropatterns on a Gold Nanostructure using Continuous-wave or Femtosecond Pulsed Near-Infrared Laser Light

Tatsuya Shoji, Junki Saitoh, Noboru Kitamura, Fumika Nagasawa, Kei Murakoshi, Hiroaki Yamauchi, Syoji Ito, Hiroshi Miyasaka, Hajime Ishihara, and Yasuyuki Tsuboi
The use of localized surface plasmons (LSPs) for highly sensitive biosensors has already been investi-gated, and they are currently being applied for the optical manipulation of small nanoparticles. The ob-jective of our work is the optical trapping of -DNA on a metallic nanostructure with femtosecond pulsed (fs) laser irradiation. Cw laser irradiation, which is generally used for plasmon excitation, not only increased the electromagnetic field intensity but also generated heat around the nanostructure, leading to the DNA becoming permanently fixed on the plasmonic substrate. Using fs laser irradiation, on the other hand, the trap-and-release of DNA was achieved by switching the fs laser irradiation on and off. This trap-and-release behavior was clearly observed using a fluorescence microscope. This technique can also be used for manipulating other biomolecules such as nucleic acids, proteins, and polysaccharides, and will prove to be a useful tool in the fabrication of biosensors.

Friday, April 19, 2013

Spatial Organization and Mechanical Properties of the Pericellular Matrix on Chondrocytes

Louis T. McLane, Patrick Chang, Anna Granqvist, Heike Boehm, Anthony Kramer, Jan Scrimgeour, Jennifer E. Curtis
A voluminous polymer coat adorns the surface of many eukaryotic cells. Although the pericellular matrix (PCM) often extends several microns from the cell surface, its macromolecular structure remains elusive. This massive cellular organelle negotiates the cell’s interaction with surrounding tissue, influencing important processes such as cell adhesion, mitosis, locomotion, molecular sequestration, and mechanotransduction. Investigations of the PCM’s architecture and function have been hampered by the difficulty of visualizing this invisible hydrated structure without disrupting its integrity. In this work, we establish several assays to noninvasively measure the ultrastructure of the PCM. Optical force probe assays show that the PCM of rat chondrocyte joint (RCJ-P) cells easily reconfigures around optically manipulated microparticles, allowing the probes to penetrate into rather than compress the matrix. We report distinct changes in forces measured from PCMs treated with exogenous aggrecan, illustrating the assay’s potential to probe proteoglycan distribution. Measurements reveal an exponentially increasing osmotic force in the PCM arising from an inherent concentration gradient. With this result, we estimate the variation of the PCM’s mesh size (correlation length) to range from ∼100 nm at the surface to 500 nm at its periphery. Quantitative particle exclusion assays confirm this prediction and show that the PCM acts like a sieve. These assays provide a much-needed tool to study PCM ultrastructure and its poorly defined but important role in fundamental cellular processes.


Radiation forces on a Rayleigh particle by highly focused partially coherent and radially polarized vortex beams

Jianhua Shu, Ziyang Chen, and Jixiong Pu
The radiation force of highly focused partially coherent and radially polarized vortex beams on a Rayleigh particle is theoretically studied. The dependence of the radiation force on coherence lengths, beam widths, topological charges of incident vortex beams, and numerical apertures of an objective is analyzed. The transverse scattering force is also investigated. It is found that the azimuthal scattering force can produce torques with respect to the optical axis if the optical tweezers are constructed by the vortex beams carrying orbit angular momentum. The direction of the torque depends on the sign of the topological charge of vortex beams, and the magnitude of the torque increases with the increase of the value of the topological charge. A Rayleigh particle can revolve around the optical axis driven by the vortex beams.

On the use of optically trapped dust particles as micro-probes in process plasmas

V. Schneider, H. Kersten

In this paper we outline the progress in the development of an optical system for particle manipulation as a plasma diagnostics method. We demonstrate basic principles and preliminary experimental results for optical trapping of microparticles in a plasma. A counter-propagating laser beam was used to trap particles in water as well as in an RF discharge. The experiments indicate that it is possible to manipulate particles, which are levitating in the plasma sheath, to obtain information on the sheath and plasma parameters.


Growth Pattern of Yeast Cells Studied under Line Optical Tweezers

S. Charrunchon, J. Limtrakul, and N. Chattham
Cell growth and division has been of scientists’ interest for over generations. Several mathematical models have been reported derived from conventional method of cell culture. Here we applied optical tweezers to guide cell division directionally. The patterns of Saccharonmyces Bayanus yeast growth was studied under 1064 nm line optical tweezers generated by time-shared multiple optical traps. Yeast growth was found following the path of the generated laser patterns in linear shape as a result of localized heating effect due to absorption at the focal point. The area of grown yeast cells as a function of time was measure through image processing. Mathematical model for the growth rate under line optical trap has been determined and discussed.

Thursday, April 18, 2013

Real-time calcium measurements of live optically trapped microorganisms

Charlie Chandsawangbhuwana, Linda Z. Shi, Qingyuan Zhu, Michael W. Berns
A system has been developed that allows for the real-time measurement of calcium dynamics in swimming sperm. Specifically, the ratiometric dye Indo-I is used as a fluorescent indicator of intracellular calcium dynamics. The dual emissions are collected by a high-sensitivity back-illuminated CCD camera coupled to a Dual-View imaging system. From the CCD, the images are sent to a custom developed algorithm which processes the images and outputs the calcium measurements in real-time. Additionally, sperm velocity and position data are processed and outputted in real-time. The velocity and position data are obtained using a separate coupled red light (>670 nm) phase contrast imaging setup that does not optically interfere with the fluorescent imaging. Using this system the effects of optical trapping on calcium dynamics was determined. Optical trapping of sperm with a decaying focused laser power of 510 mW to 3 mW over 8 seconds causes a statistically insignificant change in calcium dynamics between in-trap and out-of-trap conditions. Progesterone, a calcium activator, was added and sperm were trapped under the 8 second power decay conditions. Progesterone treated sperm has a statistically higher average calcium level than untreated sperm, but shows no statistical difference between progesterone treated in-trap and out-of-trap conditions. Trapping at 16 seconds at 510 mW without decay, which have been shown to decrease sperm motility, shows a statistical difference between baseline pre-trap and in-trap intracellular calcium levels.


Electrophoretic Retardation of Colloidal Particles in Nonpolar Liquids

Filip Strubbe, Filip Beunis, Toon Brans, Masoumeh Karvar, Wouter Woestenborghs, and Kristiaan Neyts 
We have measured the electrophoretic mobility of single, optically trapped colloidal particles, while gradually depleting the co-ions and counterions in the liquid around the particle by applying a dc voltage. This is achieved in a nonpolar liquid, where charged reverse micelles act as co-ions and counterions. By increasing the dc voltage, the mobility first increases when the concentrations of co-ions and counterions near the particle start to decrease. At sufficiently high dc voltage (around 2 V), the mobility reaches a saturation value when the co-ions and counterions are fully separated. The increase in mobility is larger when the equilibrium ionic strength is higher. The dependence of the experimental data on the equilibrium ionic strength and on the applied voltage is in good agreement with the standard theory of electrophoretic retardation, assuming that the bare particle charge remains constant. This method is useful for studying the electrophoretic retardation effect and charging mechanisms for nonpolar colloids, and it sheds light on previously unexplained particle acceleration in electronic ink devices.

Measuring three-dimensional interaction potentials using optical interference

Nassir Mojarad, Vahid Sandoghdar, and Madhavi Krishnan
We describe the application of three-dimensional (3D) scattering interferometric (iSCAT) imaging to the measurement of spatial interaction potentials for nano-objects in solution. We study electrostatically trapped gold particles in a nanofluidic device and present details on axial particle localization in the presence of a strongly reflecting interface. Our results demonstrate high-speed (~kHz) particle tracking with subnanometer localization precision in the axial and average 2.5 nm in the lateral dimension. A comparison of the measured levitation heights of trapped particles with the calculated values for traps of various geometries reveals good agreement. Our work demonstrates that iSCAT imaging delivers label-free, high-speed and accurate 3D tracking of nano-objects conducive to probing weak and long-range interaction potentials in solution.

Optically Driven Mobile Integrated Micro-Tools for a Lab-on-a-Chip

Yi-Jui Liu, Yi-Hsiung Lee, Yu-Sheng Lin, Chingfu Tsou, Patrice L. Baldeck and Chih-Lang Lin
This study proposes an optically driven complex micromachine with an Archimedes microscrew as the mechanical power, a sphere as a coupler, and three knives as the mechanical tools. The micromachine is fabricated by two-photon polymerization and is portably driven by optical tweezers. Because the microscrew can be optically trapped and rotates spontaneously, it provides driving power for the complex micro-tools. In other words, when a laser beam focuses on the micromachine, the microscrew is trapped toward the focus point and simultaneously rotates. A demonstration showed that the integrated micromachines are grasped by the optical tweezers and rotated by the Archimedes screw. The rotation efficiencies of the microrotors with and without knives are 1.9 rpm/mW and 13.5 rpm/mW, respectively. The micromachine can also be portably dragged along planed routes. Such Archimedes screw-based optically driven complex mechanical micro-tools enable rotation similar to moving machines or mixers, which could contribute to applications for a biological microfluidic chip or a lab-on-a-chip.

Enhancing the Strength of an Optical Trap by Truncation

Vanessa R. M. Rodrigues, Argha Mondal, Jayashree A. Dharmadhikari, Swapnesh Panigrahi, Deepak Mathur, Aditya K. Dharmadhikari

Optical traps (tweezers) are beginning to be used with increasing efficacy in diverse studies in the biological and biomedical sciences. We report here results of a systematic study aimed at enhancing the efficiency with which dielectric (transparent) materials can be optically trapped. Specifically, we investigate how truncation of the incident laser beam affects the strength of an optical trap in the presence of a circular aperture. Apertures of various sizes have been used by us to alter the beam radius, thereby changing the effective numerical aperture and intensity profile. We observe significant enhancement of the radial and axial trap stiffness when an aperture is used to truncate the beam compared to when no aperture was used, keeping incident laser power constant. Enhancement in trap stiffness persists even when the beam intensity profile is modulated. The possibility of applying truncation to multiple traps is explored; to this end a wire mesh is utilized to produce multiple trapping that also alters the effective numerical aperture. The use of a mesh leads to reduction in trap stiffness compared to the case when no wire mesh is used. Our findings lead to a simple-to-implement and inexpensive method of significantly enhancing optical trapping efficiency under a wide range of circumstances.

Counter-propagating dual-trap optical tweezers based on linear momentum conservation

M. Ribezzi-Crivellari, J. M. Huguet, and F. Ritort
We present a dual-trap optical tweezers setup which directly measures forces using linear momentum conservation. The setup uses a counter-propagating geometry, which allows momentum measurement on each beam separately. The experimental advantages of this setup include low drift due to all-optical manipulation, and a robust calibration (independent of the features of the trapped object or buffer medium) due to the force measurement method. Although this design does not attain the high-resolution of some co-propagating setups, we show that it can be used to perform different single molecule measurements: fluctuation-based molecular stiffness characterization at different forces and hopping experiments on molecular hairpins. Remarkably, in our setup it is possible to manipulate very short tethers (such as molecular hairpins with short handles) down to the limit where beads are almost in contact. The setup is used to illustrate a novel method for measuring the stiffness of optical traps and tethers on the basis of equilibrium force fluctuations, i.e., without the need of measuring the force vs molecular extension curve. This method is of general interest for dual trap optical tweezers setups and can be extended to setups which do not directly measure forces.

Monday, April 15, 2013

Calibration of an optical tweezer microrheometer by sequential impulse response

Matthew M. Shindel, James W. Swan, Eric M. Furst
We report a robust method for calibrating optical tweezers in any viscoelastic medium. This approach uses two coupled measurements—one from a static experiment in which a trapped particle diffuses passively within the tweezer’s harmonic potential and another from a dynamic experiment in which the trap is jumped discontinuously to a new position while the particle undergoes transient relaxation back into the minimum of the optical potential. Together, these are sufficient to determine the stiffness of the trap in a material of unknown rheology. The method is tested in a Newtonian fluid and compares favorably with other means of calibration. The calibration is also performed in a non-Newtonian fluid of which standard optical tweezer calibration methods may struggle to characterize. The correctly calibrated optical tweezer microrheometer measures the rheology of polymer solutions in agreement with macrorheological measurements.

Optical tweezers for medical diagnostics

Christopher N. LaFratta
Laser trapping by optical tweezers makes possible the spectroscopic analysis of single cells. Use of optical tweezers in conjunction with Raman spectroscopy has allowed cells to be identified as either healthy or cancerous. This combined technique is known as laser tweezers Raman spectroscopy (LTRS), or Raman tweezers. The Raman spectra of cells are complex, since the technique probes nucleic acids, proteins, and lipids; but statistical analysis of these spectra makes possible differentiation of different classes of cells. In this article the recent development of LTRS is described along with two illustrative examples for potential application in cancer diagnostics. Techniques to expand the uses of LTRS and to improve the speed of LTRS are also suggested.

Manipulation of field enhancement using tapered nanobumps with circular polarization

E. H. Khoo, I. Ahmed, and E. P. Li
Tapered nanobumps are placed on the circumference of optical vortex to manipulate the field amplitude using circular polarized light. Tapered nanobump produces stronger field enhancement due to higher charge density at the tapered end. The geometrical parameters of the tapered nanobumps are optimized to achieve highest field enhancement. The electric field is enhanced or diminished by illuminating with left or right circular polarized lightwave. Additional nanobumps are added to provide field enhancement at different parts of the vortex. This setup can be used to control the position of nanoparticles for analysis, and is useful for sensing and catalysis applications.

Applying Combined Optical Tweezers and Fluorescence Microscopy Technologies to Manipulate Cell Adhesions for Cell-to-Cell Interaction Study

Gou, X., Han, H.; Leung, A.Y.H.; Sun, D.
Cell-to-cell interactions are important for the regulation of various cell activities, such as proliferation, differentiation, and apoptosis. This paper presents an approach to studying cell-to-cell interactions at a single cell level through manipulating cell adhesions with optical tweezers. Experiments are performed on leukemia cancer cells and stromal cells to demonstrate the feasibility of this method. After the adhesion properties of leukemia cells on stromal cells are characterized, fluorescence intensity is used as a label to study the Wnt signaling pathway of leukemia cells. The activities of the Wnt signaling pathway of K562 cells on M210B4 and HS5 cells are examined based on fluorescence analysis. The reliability of the fluorescence imaging is confirmed through comparison to traditional flow cytometry analysis. The proposed approach will offer new avenues to investigate otherwise inaccessible mechanisms in cell-to-cell interactions.


Thursday, April 11, 2013

Fiber-optic trap-on-a-chip platform for probing low refractive index contrast biomaterials

Tessa M. Piñón, Alessandro R. Castelli, Linda S. Hirst, and Jay E. Sharping
Dual-beam fiber trapping is a versatile technique for manipulating microparticles. We fabricate and evaluate the performance of a compact trap-on-a-chip design and demonstrate, for what we believe is the first time, trapping of low-contrast (m<1.005) lipid vesicles in solution. Counterpropagating fibers are fixed along the chip channel, and we calibrate the trap by optically displacing polystyrene microspheres from the trap center. Measured scattering forces are ∼30–49  pN from each beam. Stable trapping and reversible deformation of lipid vesicles is demonstrated under femtonewton trapping forces. This chip has applications in probing a variety of soft biomaterials, such as biological cells, lipid membranes, and protein assemblies.

Self-diffraction of continuous laser radiation in a disperse medium with absorbing particles

O. V. Angelsky, A. Ya. Bekshaev, P. P. Maksimyak, A. P. Maksimyak, S. G. Hanson, and C. Yu. Zenkova
We study the self-action of light in a water suspension of absorbing subwavelength particles. Due to efficient accumulation of the light energy, this medium shows distinct non-linear properties even at moderate radiation power. In particular, by means of interference of two obliquely incident beams, it is possible to create controllable phase and amplitude gratings whose contrast, spatial and temporal parameters depend on the beams’ coherence and power as well as the interference geometry. The grating characteristics are investigated via the beams’ self-diffraction. The main mechanism of the grating formation is shown to be thermal, which leads to the phase grating; a weak amplitude grating also emerges due to the particles’ displacements caused by the light-induced gradient and photophoretic forces. These forces, together with the Brownian motion of the particles, are responsible for the grating dynamics and degradation. The results and approaches can be used for investigation of the thermal relaxation and kinetic processes in liquid suspensions.

Nanostructured Potential of Optical Trapping Using a Plasmonic Nanoblock Pair

Yoshito Tanaka , Shogo Kaneda , and Keiji Sasaki
We performed two-dimensional mapping of optical trapping potentials experienced by a 100 nm dielectric particle above a plasmon-resonant gold nanoblock pair with a gap of several nanometers. Our results demonstrate that the potentials have nanoscale spatial structures that reflect the near-field landscape of the nanoblock pair. When an incident polarization parallel to the pair axis is rotated by 90°, a single potential well turns into multiple potential wells separated by a distance smaller than the diffraction limit; this is associated with super-resolution optical trapping. In addition, we show that the trap stiffness can be enhanced by approximately 3 orders of magnitude compared to that with conventional far-field trapping.


Wednesday, April 10, 2013

In vivo optical trapping indicates kinesin’s stall force is reduced by dynein during intracellular transport

Benjamin H. Blehm, Trina A. Schroer, Kathleen M. Trybus, Yann R. Chemla, and Paul R. Selvin
Kinesin and dynein are fundamental components of intracellular transport, but their interactions when simultaneously present on cargos are unknown. We built an optical trap that can be calibrated in vivo during data acquisition for each individual cargo to measure forces in living cells. Comparing directional stall forces in vivo and in vitro, we found evidence that cytoplasmic dynein is active during minus- and plus-end directed motion, whereas kinesin is only active in the plus direction. In vivo, we found outward (∼plus-end) stall forces range from 2 to 7 pN, which is significantly less than the 5- to 7-pN stall force measured in vitro for single kinesin molecules. In vitro measurements on beads with kinesin-1 and dynein bound revealed a similar distribution, implying that an interaction between opposite polarity motors causes this difference. Finally, inward (∼minus-end) stalls in vivo were 2–3 pN, which is higher than the 1.1-pN stall force of a single dynein, implying multiple active dynein.

Analysis of the radiation force and torque exerted on a chiral sphere by a Gaussian beam

Qing-Chao Shang, Zhen-Sen Wu, Tan Qu, Zheng-Jun Li, Lu Bai, and Lei Gong

Under the framework of generalized Lorenz-Mie theory, we calculate the radiation force and torque exerted on a chiral sphere by a Gaussian beam. The theory and codes for axial radiation force are verified when the chiral sphere degenerates into an isotropic sphere. We discuss the influence of a chirality parameter on the radiation force and torque. Linearly and circularly polarized incident Gaussian beams are considered, and the corresponding radiation forces and torques are compared and analyzed. The polarization of the incident beam considerably influences radiation force of a chiral sphere. In trapping a chiral sphere, therefore, the polarization of incident beams should be chosen in accordance with the chirality. Unlike polarization, variation of chirality slightly affects radiation torque, except when the imaginary part of the chirality parameter is considered.

Calibration of nonspherical particles in optical tweezers using only position measurement

Ann A. M. Bui, Alexander B. Stilgoe, Timo A. Nieminen, and Halina Rubinsztein-Dunlop
Nonspherical probe particles are an attractive choice for optically-trapped scanning probe microscopy. We show that it is possible to calibrate a trap with a nonspherical particle using only position measurements, without requiring measurement of orientation, using a pseudopotential based on the position occupation probability. It is not necessary to assume the force is linear with displacement.


Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level

Carina Monico, Marco Capitanio, Gionata Belcastro, Francesco Vanzi and Francesco S. Pavone

The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes, critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety of methods in biology. A large interest in these processes stems from the faster-than-diffusion association rates, explained in current models by a combination of 3D and 1D diffusion. Here, we present a review of the single-molecule approaches at the forefront of the study of protein-DNA interaction dynamics and target search in vitro and in vivo. Flow stretch, optical and magnetic manipulation, single fluorophore detection and localization as well as combinations of different methods are described and the results obtained with these techniques are discussed in the framework of the current facilitated diffusion model.

Optical lock-in particle tracking in optical tweezers

Michael A Taylor, Joachim Knittel, and Warwick P Bowen
We demonstrate a lock-in particle tracking scheme in optical tweezers based on stroboscopic modulation of an illuminating optical field. This scheme is found to evade low frequency noise sources while otherwise producing an equivalent position measurement to continuous measurement. This was demonstrated to yield up to 20 dB of noise suppression at both low frequencies (< 1 kHz), where low frequency electronic noise was significant, and around 630 kHz where laser relaxation oscillations introduced laser noise. The setup is simple, and compatible with any trapping optics.

Tuesday, April 9, 2013

Tailorable three-dimensional distribution of laser foci based on customized fractal zone plates

S H Tao, B C Yang, H Xia and W X Yu
There is high demand for a tailorable three-dimensional (3D) distribution of focused laser beams for simultaneous optical manipulation of multiple particles separately distributed in 3D space. In this letter, accurate control of the 3D distribution of laser beam foci is demonstrated with an array of customized fractal zone plates (FZPs). The FZPs are designed with a fractional number of fractal segments, so the focal lengths of the foci can be finely tailored. The unique focusing properties of the customized FZPs are investigated with both simulations and experiments. The FZP beams are also found to possess the self-reconstruction property, which would be useful for constructing 3D optical tweezers.


Nonequilibrium Distributions and Hydrodynamic Coupling Distort the Measurement of Nanoscale Forces near Interfaces

James W. Swan, Eric M. Furst
We calculate the displacement of a single spherical particle from the minimum of a harmonic well positioned near a plane wall and immersed in a uniform flow. A failure to account for the fluctuations in particle position orthogonal to the plane (leading to fluctuations in hydrodynamic drag) results in large discrepancies, with the naive displacement calculated by assuming no fluctuations in the balance of forces. The chief criterion for neglecting such fluctuations is that the stiffness of the harmonic potential exceeds the thermal stresses on the particle by at least two orders of magnitude. For micrometer-diameter particles typically employed in force spectroscopy of DNA, macromolecules, and molecular motors, this can lead to errors of up to 100% in the measured properties. The Supporting Material to the article provides an implementation of this model intended to fit experimental measurements for the stiffness of the harmonic potential constraining the particle.

Frequency-dependent Cell Death by Optical Tweezers Manipulation

K.S. Ng, Z.L. Zhou, A.H.W. Ngan

Optical tweezers were used to scan individual Chronic Myelogenous Leukemia cells to determine if the cell death depends on the scanning conditions. Although increasing the scanning frequency or amplitude means greater force applied to the cells, their effects on cell death are not a simple increasing trend, as observed in the optical microscopy. Indeed, cell death sharply increased at particular screening frequencies and amplitudes, whereas other frequencies or amplitudes were less detrimental. These results suggest that cell damage was more sensitive to certain scanning conditions, rather than simply high applied forces.


Localized surface plasmon-enhanced propulsion of gold nanospheres

Ying Li and Yanjun Hu

We experimentally demonstrated the enhanced propulsion of 250 nm gold nanospheres using an optical nanofiber decorated with five gold nanoparticles. By tuning the input laser wavelength to 808 nm, the enhanced propulsion phenomenon occurred due to the excitation of local surface plasmon resonance (LSPR) of the gold nanoparticles. Simulated results indicate considerably enhanced optical scattering force on the gold nanospheres provided by LSPR, which lead to enhanced propulsion velocity. The velocity was measured to be about 10 times larger for the LSPR of gold nanoparticles than the conventional evanescent field around the nanofiber.

Optical trapping of nanoparticles by ultrashort laser pulses

Usman, Anwar; Chiang, Wei-Yi; Masuhara, Hiroshi
Optical trapping with continuous-wave lasers has been a fascinating field in the optical manipulation. It has become a powerful tool for manipulating micrometer-sized objects, and has been widely applied in physics, chemistry, biology, material, and colloidal science. Replacing the continuous-wave- with pulsed-mode laser in optical trapping has already revealed some novel phenomena, including the stable trap, modifiable trapping positions, and controllable directional ejections of particles in nanometer scales. Due to two distinctive features; impulsive peak powers and relaxation time between consecutive pulses, the optical trapping with the laser pulses has been demonstrated to have some advantages over conventional continuous-wave lasers, particularly when the particles are within Rayleigh approximation. This would open unprecedented opportunities in both fundamental science and application. This Review summarizes recent advances in the optical trapping with laser pulses and discusses the electromagnetic formulations and physical interpretations of the new phenomena. Its aim is rather to show how beautiful and promising this field will be, and to encourage the in-depth study of this field.

Optical micromanipulation of active cells with minimal perturbations: direct and indirect pushing

Chenlu Wang ; Sagar Chowdhury ; Satyandra K. Gupta ; Wolfgang Losert
The challenge to wide application of optical tweezers in biological micromanipulation is the photodamage caused by high-intensity laser exposure to the manipulated living systems. While direct exposure to infrared lasers is less likely to kill cells, it can affect cell behavior and signaling. Pushing cells with optically trapped objects has been introduced as a less invasive alternative, but the technique includes some exposure of the biological object to parts of the optical tweezer beam. To keep the cells farther away from the laser, we introduce an indirect pushing-based technique for noninvasive manipulation of sensitive cells. We compare how cells respond to three manipulation approaches: direct manipulation, pushing, and indirect pushing. We find that indirect manipulation techniques lessen the impact of manipulation on cell behavior. Cell survival increases, as does the ability of cells to maintain shape and wiggle. Our experiments also demonstrate that indirect pushing allows cell–cell contacts to be formed in a controllable way, while retaining the ability of cells to change shape and move.


Sunday, April 7, 2013

Red cell investigations: Art and artefacts

Giampaolo Minetti, Stephane Egée, Daniel Mörsdorf, Patrick Steffen, Asya Makhro, Cesare Achilli, Annarita Ciana, Jue Wang, Guillaume Bouyer, Ingolf Bernhardt, Christian Wagner, Serge Thomas, Anna Bogdanova, Lars Kaestner
Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology.
Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards.
Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy.

From mechanical folding trajectories to intrinsic energy landscapes of biopolymers

Michael Hinczewski, J. Christof M. Gebhardt, Matthias Rief, and D. Thirumalai
In single-molecule laser optical tweezer (LOT) pulling experiments, a protein or RNA is juxtaposed between DNA handles that are attached to beads in optical traps. The LOT generates folding trajectories under force in terms of time-dependent changes in the distance between the beads. How to construct the full intrinsic folding landscape (without the handles and beads) from the measured time series is a major unsolved problem. By using rigorous theoretical methods—which account for fluctuations of the DNA handles, rotation of the optical beads, variations in applied tension due to finite trap stiffness, as well as environmental noise and limited bandwidth of the apparatus—we provide a tractable method to derive intrinsic free-energy profiles. We validate the method by showing that the exactly calculable intrinsic free-energy profile for a generalized Rouse model, which mimics the two-state behavior in nucleic acid hairpins, can be accurately extracted from simulated time series in a LOT setup regardless of the stiffness of the handles. We next apply the approach to trajectories from coarse-grained LOT molecular simulations of a coiled-coil protein based on the GCN4 leucine zipper and obtain a free-energy landscape that is in quantitative agreement with simulations performed without the beads and handles. Finally, we extract the intrinsic free-energy landscape from experimental LOT measurements for the leucine zipper.

Denaturation transition of stretched DNA

Andreas Hanke
In the last two decades, single-molecule force measurements using optical and magnetic tweezers and atomic force spectroscopy have dramatically expanded our knowledge of nucleic acids and proteins. These techniques characterize the force on a biomolecule required to produce a given molecular extension. When stretching long DNA molecules, the observed force–extension relationship exhibits a characteristic plateau at approximately 65 pN where the DNA may be extended to almost twice its B-DNA length with almost no increase in force. In the present review, I describe this transition in terms of the Poland–Scheraga model and summarize recent related studies.

Dynamic trapping and manipulation of biological cells with optical tweezers

Xiang Li, Chien Chern Cheah, Songyu Hu, Dong Sun
Current control techniques for optical tweezers work only when the cell is located in a small neighbourhood around the centroid of the focused light beam. Therefore, the optical trapping fails when the cell is initially located far away from the laser beam or escapes from the optical trap during manipulation. In addition, the position of the laser beam is treated as the control input in existing optical tweezers systems and an open-loop controller is designed to move the laser source. In this paper, we propose a new robotic manipulation technique for optical tweezers that integrates automatic trapping and manipulation of biological cells into a single method. Instead of using open-loop control of the position of laser source as assumed in the literature, a closed-loop dynamic control method is formulated and solved in this paper. We provide a theoretical framework that bridges the gap between traditional robotic manipulation techniques and optical manipulation techniques of cells. The proposed controller allows the transition from trapping to manipulation without any hard switching from one controller to another. Simulation and experimental results are presented to illustrate the performance of the proposed controller.

Saturday, April 6, 2013

FBAR Syndapin 1 recognizes and stabilizes highly curved tubular membranes in a concentration dependent manner

Pradeep Ramesh, Younes F. Baroji, S. Nader S. Reihani, Dimitrios Stamou, Lene B. Oddershede & Poul Martin Bendix
Syndapin 1 FBAR, a member of the Bin-amphiphysin-Rvs (BAR) domain protein family, is known to induce membrane curvature and is an essential component in biological processes like endocytosis and formation and growth of neurites. We quantify the curvature sensing of FBAR on reconstituted porcine brain lipid vesicles and show that it senses membrane curvature at low density whereas it induces and reinforces tube stiffness at higher density. FBAR strongly up-concentrates on the high curvature tubes pulled out of Giant Unilamellar lipid Vesicles (GUVs), this sorting behavior is strongly amplified at low protein densities. Interestingly, FBAR from syndapin 1 has a large affinity for tubular membranes with curvatures larger than its own intrinsic concave curvature. Finally, we studied the effect of FBAR on membrane relaxation kinetics with high temporal resolution and found that the protein increases relaxation time of the tube holding force in a density-dependent fashion.

Novel single-cell functional analysis of red blood cells using laser tweezers raman spectroscopy: application for sickle cell disease

Rui Liu, Ziliang Mao, Dennis L. Matthews, Chin-Shang Li, James W. Chan, Noriko Satake
Laser tweezers Raman spectroscopy was used to characterize the oxygenation response of single normal adult, sickle, and cord blood red blood cells (RBCs) to an applied mechanical force. Individual cells were subjected to different forces by varying the laser power of a single-beam optical trap, and the intensities of several oxygenation-specific Raman spectral peaks were monitored to determine the oxygenation state of the cells. For all three cell types, an increase in laser power (or mechanical force) induced a greater deoxygenation of the cell. However, sickle RBCs deoxygenated more readily than normal RBCs when subjected to the same optical forces. Conversely, cord blood RBCs were able to maintain their oxygenation better than normal RBCs. These results suggest that differences in the chemical or mechanical properties of fetal, normal, and sickle cells affect the degree to which applied mechanical forces can deoxygenate the cell. Populations of normal, sickle, and cord RBCs were identified and discriminated based on this mechanochemical phenomenon. This study demonstrates the potential application of laser tweezers Raman spectroscopy as a single-cell, label-free analytical tool to characterize the functional (e.g., mechanical deformability, oxygen binding) properties of normal and diseased RBCs.

Monday, April 1, 2013

The specificity of the interaction between αB-crystallin and desmin filaments and its impact on filament aggregation and cell viability

Jayne L. Elliott, Ming Der Perng, Alan R. Prescott, Karin A. Jansen, Gijsje H. Koenderink and Roy A. Quinlan
CRYAB (αB-crystallin) is expressed in many tissues and yet the R120G mutation in CRYAB causes tissue-specific pathologies, namely cardiomyopathy and cataract. Here, we present evidence to demonstrate that there is a specific functional interaction of CRYAB with desmin intermediate filaments that predisposes myocytes to disease caused by the R120G mutation. We use a variety of biochemical and biophysical techniques to show that plant, animal and ascidian small heat-shock proteins (sHSPs) can interact with intermediate filaments. Nevertheless, the mutation R120G in CRYAB does specifically change that interaction when compared with equivalent substitutions in HSP27 (R140G) and into the Caenorhabditis elegans HSP16.2 (R95G). By transient transfection, we show that R120G CRYAB specifically promotes intermediate filament aggregation in MCF7 cells. The transient transfection of R120G CRYAB alone has no significant effect upon cell viability, although bundling of the endogenous intermediate filament network occurs and the mitochondria are concentrated into the perinuclear region. The combination of R120G CRYAB co-transfected with wild-type desmin, however, causes a significant reduction in cell viability. Therefore, we suggest that while there is an innate ability of sHSPs to interact with and to bind to intermediate filaments, it is the specific combination of desmin and CRYAB that compromises cell viability and this is potentially the key to the muscle pathology caused by the R120G CRYAB.

Step Detection in Single-Molecule Real Time Trajectories Embedded in Correlated Noise

Srikesh G. Arunajadai, Wei Cheng
Single-molecule real time trajectories are embedded in high noise. To extract kinetic or dynamic information of the molecules from these trajectories often requires idealization of the data in steps and dwells. One major premise behind the existing single-molecule data analysis algorithms is the Gaussian ‘white’ noise, which displays no correlation in time and whose amplitude is independent on data sampling frequency. This so-called ‘white’ noise is widely assumed but its validity has not been critically evaluated. We show that correlated noise exists in single-molecule real time trajectories collected from optical tweezers. The assumption of white noise during analysis of these data can lead to serious over- or underestimation of the number of steps depending on the algorithms employed. We present a statistical method that quantitatively evaluates the structure of the underlying noise, takes the noise structure into account, and identifies steps and dwells in a single-molecule trajectory. Unlike existing data analysis algorithms, this method uses Generalized Least Squares (GLS) to detect steps and dwells. Under the GLS framework, the optimal number of steps is chosen using model selection criteria such as Bayesian Information Criterion (BIC). Comparison with existing step detection algorithms showed that this GLS method can detect step locations with highest accuracy in the presence of correlated noise. Because this method is automated, and directly works with high bandwidth data without pre-filtering or assumption of Gaussian noise, it may be broadly useful for analysis of single-molecule real time trajectories.

Measurement of Monolayer Viscosity Using Noncontact Microrheology

Roie Shlomovitz, Arthur A. Evans, Thomas Boatwright, Michael Dennin, and Alex J. Levine
Microrheological studies of phospholipid monolayers, bilayers, and other Langmuir monolayer systems are traditionally performed by observing the thermal fluctuations of tracers attached to the membrane or interface. Measurements of this type obtain surface moduli that are orders of magnitude different from those obtained using macroscopic or active techniques. These large discrepancies can result from uncertainties in the tracer’s coupling to the monolayer or the local disruption of the monolayer by the tracer. To avoid such problems, we perform a microrheological experiment with the tracer particle placed at a known depth beneath the monolayer; this avoids the issues mentioned at the cost of generating a weaker, purely hydrodynamic coupling between the tracer and the monolayer. We calculate the appropriate response functions for this submerged particle microrheology and demonstrate the technique on three model monolayer systems.

Effective heating to several thousand kelvins of an optically trapped sphere in a liquid

Ignacio A. Martínez, Édgar Roldán, Juan M. R. Parrondo, and Dmitri Petrov
The cooling of the center of mass motion of optically trapped microspheres by feedback stabilization is advancing rapidly, and cooling below several millikelvin is possible. Such a controllable attenuation of the motion is an important step towards new experiments in different areas of physics. In this study we suggest going in the opposite direction, in controlling the motion of optically trapped spheres, namely, to increase the amplitude of their Brownian fluctuations. We show that the effective kinetic temperature of a Brownian particle may achieve 3000 K when an additional external random force is applied to the sphere. We demonstrate experimentally how the temperature increase affects the histogram of the position of the Brownian particle, its power spectral density, its response to an external perturbation, and the statistics of the Kramers transitions in a double-well potential. Effects related to the nonideal character of the white noise generated experimentally are also analyzed. This experimental technique allows tuning and controlling the kinetic temperature of the sphere with millisecond resolution over a wide range and along a single spatial direction, and has considerable potential for the study of thermodynamic processes at the microscopic scale.