Thursday, December 19, 2013

High-Speed Force Spectroscopy Unfolds Titin at the Velocity of Molecular Dynamics Simulations

Felix Rico, Laura Gonzalez, Ignacio Casuso, Manel Puig-Vidal, Simon Scheuring

The mechanical unfolding of the muscle protein titin by atomic force microscopy was a landmark in our understanding of single-biomolecule mechanics. Molecular dynamics simulations offered atomic-level descriptions of the forced unfolding. However, experiment and simulation could not be directly compared because they differed in pulling velocity by orders of magnitude. We have developed high-speed force spectroscopy to unfold titin at velocities reached by simulation (~4 millimeters per second). We found that a small β-strand pair of an immunoglobulin domain dynamically unfolds and refolds, buffering pulling forces up to ~100 piconewtons. The distance to the unfolding transition barrier is larger than previously estimated but is in better agreement with atomistic predictions. The ability to directly compare experiment and simulation is likely to be important in studies of biomechanical processes.


Particle tracking by full-field complex wavefront subtraction in digital holography microscopy

Lisa Miccio, Pasquale Memmolo , Francesco Merola, Sabato Fusco, Antonio Paciello, Maurizio Ventre, Paolo Netti and Pietro Ferraro

3D tracking of micro-objects, based on digital holography, is proposed through the analysis of the complex wavefront of the light scattered by the micro-samples. Exploiting the advantages of the off-axis full-field holographic interferometry, tracking of multiple objects is achieved by a direct wavefront analysis at the focal plane overcoming the limitation of the conventional Back Focal Plane interferometry in which one object at time can be tracked. Furthermore, the method proposed and demonstrated here is a step-forward also in respect to other holographic tracking tools. The approach is tested in two experiments, one for investigating Brownian motion of particles trapped by holographic optical tweezers, while second is related to cells motility in 3D collagen matric thus showing its usefulness for lab-on-chip systems in typical bio-assay testing.


Measurements of the Sensitivity of Aerosol Hygroscopicity and the κ Parameter to the O/C Ratio

Andrew M. J. Rickards , Rachael E. H. Miles , James F. Davies , Frances H. Marshall , and Jonathan P. Reid

We report measurements of the subsaturated hygroscopic growth of aerosol particles composed of single organic components of varying oxygen-to-carbon ratio up to relative humidities approaching saturation using the techniques of aerosol optical tweezers and an electrodynamic balance. The variation in the hygroscopicity parameter κ between compounds of even the same O/C ratio is found to be significant with, for example, a range in κ values from 0.12 to 0.38 for compounds with an O/C of 1. The measurements are compared with a review of all of the available literature data for which both the κ value and O/C ratio are reported, and a new parametrization is determined. Critical supersaturations predicted using this parametrization yield values that have associated uncertainties that are comparable to typical uncertainties in experimental measurements of critical supersaturations. However, the systematic variability between κ parametrizations determined from different studies remains large, consistent with the O/C ratio providing only an approximate guide to aerosol hygroscopicity and reflecting significant variations for aerosols of different chemical functionality, composition, and oxidation history.


Use of topological defects as templates to direct assembly of colloidal particles at nematic interfaces

Mohamed Amine Gharbi, Maurizio Nobili, Christophe Blanc

In this work, we experimentally investigate the ability of topological defects to guide interfacial assembly of spherical particles with homeotropic anchoring confined to nematic interfaces. We propose two different systems: In the first one, particles are trapped at an air/nematic interface where they spontaneously form various 2D patterns. We demonstrate that the phase transition between these patterns can be controlled by defects formed in the nematic bulk. In the second system, we explore the behavior of particles at the surface of bipolar nematic drops. We found that particles assemble into linear chains and interact with surface defects at the North and South poles of the drop, giving rise to the formation of star structures in a self-assembly process. We detail the mechanism that guides the behavior of particles and discuss the role of defects in the formation of the observed patterns.


Radiation pressure efficiency measurements of nanoparticle coated microspheres

Soo Y. Kim, Joseph D. Taylor, Harold D. Ladouceur Sean J. Hart and Alex Terray

Experimental measurements of the radiation pressure efficiency (Qpr ) for several microparticles have been compared to theoretical calculations extrapolated from the Bohren-Huffman code for Mie scattering of coated particles. An increased shift of the Qpr parameter was observed for 2 μm SiO2 core particles coated with nanoparticles of higher refractive indices. Coatings of 14 nm melamine particles were found to increase the Qpr parameter 135 times over similar coatings using SiO2 particles of the same size. While a coating of 100 nm polystyrene particles also showed a significant increase, they did not agree well with theoretical values. It is hypothesized that other factors such as increased scatter, drag, and finite coating coverage are no longer negligible for coatings using nanoparticles in this size regime.


Quantum noise in optical tweezers

Michael A Taylor and Warwick P Bowen

Quantum enhanced sensitivity in optical tweezers based particle tracking was recently demonstrated. This has provided the necessary tool for quantum metrology to play an important role in biological measurements. Here we introduce the basic theory relevant to such optical tweezers experiments, and overview the significance of sub-shot noise limited sensitivity to practical experiments. In particular, biophysical experiments are subject to optical power constraints, which therefore limits the absolute sensitivity which is classically achievable. Quantum enhanced particle tracking can overcome this limit, and is therefore likely to play an important role in such biophysical experiments in the near future.


Focused plasmonic trapping of metallic particles

Changjun Min, Zhe Shen, Junfeng Shen, Yuquan Zhang, Hui Fang, Guanghui Yuan, Luping Du, Siwei Zhu, Ting Lei & Xiaocong Yuan

Scattering forces in focused light beams push away metallic particles. Thus, trapping metallic particles with conventional optical tweezers, especially those of Mie particle size, is difficult. Here we investigate a mechanism by which metallic particles are attracted and trapped by plasmonic tweezers when surface plasmons are excited and focused by a radially polarized beam in a high-numerical-aperture microscopic configuration. This contrasts the repulsion exerted in optical tweezers with the same configuration. We believe that different types of forces exerted on particles are responsible for this contrary trapping behaviour. Further, trapping with plasmonic tweezers is found not to be due to a gradient force balancing an opposing scattering force but results from the sum of both gradient and scattering forces acting in the same direction established by the strong coupling between the metallic particle and the highly focused plasmonic field. Theoretical analysis and simulations yield good agreement with experimental results.


Wednesday, December 18, 2013

Single and dual fiber nano-tip optical tweezers: trapping and analysis

Jean-Baptiste Decombe, Serge Huant, and Jochen Fick

An original optical tweezers using one or two chemically etched fiber nano-tips is developed. We demonstrate optical trapping of 1 micrometer polystyrene spheres at optical powers down to 2 mW. Harmonic trap potentials were found in the case of dual fiber tweezers by analyzing the trapped particle position fluctuations. The trap stiffness was deduced using three different models. Consistent values of up to 1 fN/nm were found. The stiffness linearly decreases with decreasing light intensity and increasing fiber tip-to-tip distance.


Monday, December 16, 2013

Optimal Hydrodynamic Synchronization of Colloidal Rotors

Jurij Kotar, Luke Debono, Nicolas Bruot, Stuart Box, David Phillips, Stephen Simpson, Simon Hanna, and Pietro Cicuta

Synchronization of driven oscillators is a key aspect of flow generation in artificial and biological filaments such as cilia. Previous theoretical and numerical studies have considered the “rotor” model of a cilium in which the filament is coarse grained into a colloidal sphere driven with a given force law along a predefined trajectory to represent the oscillating motion of the cilium. These studies pointed to the importance of two factors in the emergence of synchronization: the modulation of the driving force around the orbit and the deformability of the trajectory. In this work it is shown via experiments, supported by numerical simulations and theory, that both of these factors are important and can be combined to produce strong synchronization (within a few cycles) even in the presence of thermal noise.


Timescales of water transport in viscous aerosol: measurements on sub-micron particles and dependence on conditioning history

Jessica W. Lu, Andrew M. J. Rickards, Jim S. Walker, Kerry J. Knox, Rachael E. H. Miles, Jonathan P. Reid and Ruth Signorell

Evaporation studies of single aqueous sucrose aerosol particles as a function of relative humidity (RH) are presented for coarse and fine mode particles down into the submicron size range (600 nm < r < 3.0 μm). These sucrose particles serve as a proxy for biogenic secondary organic aerosols that have been shown to exist, under ambient conditions, in an ultraviscous glassy state, which can affect the kinetics of water mass transport within the bulk phase and hinder particle response to changes in the gas phase water content. A counter-propagating Bessel beams (CPBBs) optical trapping setup is employed to monitor the real-time change in the particle radius with RH decreasing from 75% to 5%. The slow-down of the size change upon each RH step and the deviation from the theoretical equilibrium hygroscopic growth curve indicate the onset of glassy behavior in the RH range of 10–40%. Size-dependent effects were not observed within the uncertainty of the measurements. The influence of the drying time below the glass transition RH on the timescale of subsequent water condensation and re-equilibration for sucrose particles is explored by optical tweezers measurements of micron-sized particles (3 μm < r < 6 μm). The timescale for water condensation and re-equilibration is shown to increase with increasing drying time, i.e. the time over which a viscous particle is dried below 5% RH. These studies demonstrate the importance of the history of the particle conditioning on subsequent water condensation and re-equilibration dynamics of ultraviscous and glassy aerosol particles.


Differential contribution of basic residues to HIV-1 nucleocapsid protein’s nucleic acid chaperone function and retroviral replication

Hao Wu, Mithun Mitra, M. Nabuan Naufer, Micah J. McCauley, Robert J. Gorelick, Ioulia Rouzina, Karin Musier-Forsyth and Mark C. Williams

The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC’s NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC’s aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity.


A high-speed vertical optical trap for the mechanical testing of living cells at piconewton forces

Kai Bodensiek, Weixing Li, Paula Sánchez, Schanila Nawaz and Iwan A. T. Schaap

Although atomic force microscopy is often the method of choice to probe the mechanical response of (sub)micrometer sized biomaterials, the lowest force that can be reliably controlled is limited to ≈0.1 nN. For soft biological samples, like cells, such forces can already lead to a strain large enough to enter the non-elastic deformation regime. To be able to investigate the response of single cells at lower forces we developed a vertical optical trap. The force can be controlled down to single piconewtons and most of the advantages of atomic force microscopy are maintained, such as the symmetrical application of forces at a wide range of loading rates. Typical consequences of moving the focus in the vertical direction, like the interferometric effect between the bead and the coverslip and a shift of focus, were quantified and found to have negligible effects on our measurements. With a fast responding force feedback loop we can achieve deformation rates as high as 50 μm/s, which allow the investigation of the elastic and viscous components of very soft samples. The potential of the vertical optical trap is demonstrated by measuring the linearity of the response of single cells at very low forces and a high bandwidth of deformation rates.


Holographic optical tweezers combined with back-focal-plane displacement detection

Ferran Marsà, Arnau Farré, Estela Martín-Badosa, and Mario Montes-Usategui

A major problem with holographic optical tweezers (HOTs) is their incompatibility with laser-based position detection methods, such as back-focal-plane interferometry (BFPI). The alternatives generally used with HOTs, like high-speed video tracking, do not offer the same spatial and temporal bandwidths. This has limited the use of this technique in precise quantitative experiments. In this paper, we present an optical trap design that combines digital holography and back-focal-plane displacement detection. We show that, with a particularly simple setup, it is possible to generate a set of multiple holographic traps and an additional static non-holographic trap with orthogonal polarizations and that they can be, therefore, easily separated for measuring positions and forces with the high positional and temporal resolutions of laser-based detection. We prove that measurements from both polarizations contain less than 1% crosstalk and that traps in our setup are harmonic within the typical range. We further tested the instrument in a DNA stretching experiment and we discuss an interesting property of this configuration: the small drift of the differential signal between traps.


Single Molecule Applications of Quantum Dots

Thomas E. Rasmussen, Liselotte Jauffred, Jonathan Brewer, Stefan Vogel, Esben R. Torbensen, B. Christoffer Lagerholm, Lene Oddershede, Eva C. Arnspang

Fluorescent nanocrystals composed of semiconductor materials were first introduced for biological applications in the late 1990s. The focus of this review is to give a brief survey of biological applications of quantum dots (QDs) at the single QD sensitivity level. These are described as follows: 1) QD blinking and bleaching statistics, 2) the use of QDs in high speed single particle tracking with a special focus on how to design the biofunctional coatings of QDs which enable specific targeting to single proteins or lipids of interest, 3) a hybrid lipid-DNA analogue binding QDs which allows for tracking single lipids in lipid bilayers, 4) two-photon fluorescence correlation spectroscopy of QDs and 5) optical trapping and excitation of single QDs. In all of these applications, the focus is on the single particle sensitivity level of QDs. The high applicability of QDs in live cell imaging experiments held together with the prospects in localization microscopy and single molecule manipulation experiments gave QDs a promising future in single molecule research.


Determining the Specificity of Monoclonal Antibody HPT-101 to Tau-Peptides with Optical Tweezers

Tim Stangner, Carolin Wagner, David Singer, Stefano Angioletti-Uberti, Christof Gutsche, Joachim Dzubiella, Ralf Hoffmann, and Friedrich Kremer

Optical tweezers-assisted dynamic force spectroscopy is employed to investigate specific receptor–ligand interactions on the level of single binding events. In particular, we analyze binding of the phosphorylation-specific monoclonal antibody (mAb) HPT-101 to synthetic tau-peptides with two potential phosphorylation sites (Thr231 and Ser235), being the most probable markers for Alzheimer’s disease. Whereas the typical interpretation of enzyme-linked immunosorbent assay (ELISA) suggests that this monoclonal antibody binds exclusively to the double-phosphorylated tau-peptide, we show here by DFS that the specificity of only mAb HPT-101 is apparent. In fact, binding occurs also to each sort of monophosphorylated peptide. Therefore, we characterize the unbinding process by analyzing the measured rupture force distributions, from which the lifetime of the bond without force τ0, its characteristic length xts, and the free energy of activation ΔG are extracted for the three mAb/peptide combinations. This information is used to build a simple theoretical model to predict features of the unbinding process for the double-phosphorylated peptide purely based on data on the monophosphorylated ones. Finally, we introduce a method to combine binding and unbinding measurements to estimate the relative affinity of the bonds. The values obtained for this quantity are in accordance with ELISA, showing how DFS can offer important insights about the dynamic binding process that are not accessible with this common and widespread assay.


Friday, December 13, 2013

Theory and experiment on particle trapping and manipulation via optothermally generated bubbles

Chenglong Zhao, Yuliang Xie, Zhangming Mao, Yanhui Zhao, Joseph Rufo, Shikuan Yang, Feng Guo, John D. Mai and Tony Jun Huang

We present a theoretical analysis and experimental demonstration of particle trapping and manipulation around optothermally generated bubbles. We show that a particle located within 500 μm of a surface bubble can be attracted towards a bubble by drag force resulting from a convective flow. Once the particle comes in contact with the bubble's surface, a balance between surface tension forces and pressure forces traps the particle on the bubble surface, allowing the particle to move with the bubble without detaching. The proposed mechanism is confirmed by computational fluid dynamics simulations, force calculations, and experiments. Based on this mechanism, we experimentally demonstrated a novel approach for manipulating microparticles via optothermally generated bubbles. Using this approach, randomly distributed microparticles were effectively collected and carried to predefined locations. Single particles were also manipulated along prescribed trajectories. This bubble-based particle trapping and manipulation technique can be useful in applications such as micro assembly, particle concentration, and high-precision particle separation.


Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein

Kathy R. Chaurasiya, Micah J. McCauley, Wei Wang, Dominic F. Qualley, Tiyun Wu, Shingo Kitamura, Hylkje Geertsema, Denise S. B. Chan, Amber Hertz, Yasumasa Iwatani, Judith G. Levin, Karin Musier-Forsyth, Ioulia Rouzina & Mark C. Williams

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalysed DNA elongation. Here, we show that APOBEC3G initially binds ssDNA with rapid on–off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription.


Raman spectroscopic investigations on optical trap induced deoxygenation of red blood cells

Sunita Ahlawat, Nitin Kumar, Raktim Dasgupta, Ravi Shanker Verma, Abha Uppal and Pradeep Kumar Gupta

Raman spectroscopic investigations on the oxygenation status of optically trapped red blood cells show that the cellular site in the trap beam is more deoxygenated compared to the rest of the cell, and the level of deoxygenation increases with an increase in the trap beam power. These observations and the changes in the Raman spectrum of hemoglobin solution as a function of the trapping beam power suggest that observed deoxygenation may be due to photodissociation of oxygen from hemoglobin at increased trapping power.


White Light Trapping Using Supercontinuum Generation Spectra in a Lead-Silicate Fibre Taper

Pengfei Wang; Lee, T. ; Ming Ding ; Zhenggang Lian ; Xian Feng ; Youqiao Ma ; Lin Bo ; Qiang Wu ; Semenova, Y. ; Wei Loh ; Farrell, G. ; Brambilla, G.

We experimentally investigate white light optical trapping by generating a supercontinuum in a lead silicate fibre pumped by femtosecond pulses from a Ti:Sapphire laser near the zero-dispersion wavelength of 1030 nm, before confining the light using a microfibre half taper with a final tip diameter of 75 nm. Due to the high intensity gradient at the output, robust optical trapping is possible, as demonstrated for individual yeast cells using an average pumping power of 100 mW.


Observer-Based Optical Manipulation of Biological Cells With Robotic Tweezers

Cheah, C.C. ; Li, X. ; Yan, X. ; Sun, D.

While several automatic manipulation techniques have recently been developed for optical tweezer systems, the measurement of the velocity of cell is required and the interaction between the cell and the manipulator of laser source is usually ignored in these formulations. Although the position of cell can be measured by using a camera, the velocity of cell is not measurable and usually estimated by differentiating the position of cell, which amplifies noises and may induce chattering of the system. In addition, it is also assumed in existing methods that the image Jacobian matrix from the Cartesian space to image space of the camera is exactly known. In the presence of estimation errors or variations of depth information between the camera and the cell, it is not certain whether the stability of the system could still be ensured. In this paper, vision-based observer techniques are proposed for optical manipulation to estimate the velocity of cell. Using the proposed observer techniques, tracking control strategies are developed to manipulate biological cells with different Reynolds numbers, which do not require camera calibration and measurement of the velocity of cell. The control methods are based on the dynamic formulation where the laser source is controlled by the closed-loop robotic manipulation technique. The stability is analyzed using Lyapunov-like analysis. Simulation and experimental results are presented to illustrate the performance of the proposed cell manipulation methods.


Living cell manipulation in a microfluidic device by femtosecond optical tweezers

Yan Li, Zhongyi Guo, Shiliang Qu

We have realized cell sorting and manipulation in a fabricated microfluidic device by a self-constructed femtosecond optical tweezer efficaciously. The used microfluidic device with two micro-pools inside silica glass is fabricated by water-assisted femtosecond laser ablation and subsequent heat treatment. After the heat treatment, the diameter of the fabricated microchannels could be reduced significantly and the internal surface of the device could also be made much smoother comparatively, which was crucial for the subsequent experiments of living cell manipulation and cell sorting by using femtosecond optical tweezers because of the optical beam quality required. Our experimental results show that we can manipulate cells very easily by our self-constructed femtosecond optical tweezer, which demonstrates that the incorporation of the microfluidic device and the femtosecond optical tweezer is accessible and practical for the micromanipulation experiments, such as cell sorting and manipulation.


Thursday, December 12, 2013

Hydrodynamic effects in the measurement of interparticle forces in nematic colloids

Kuniyoshi Izaki and Yasuyuki Kimura

We propose improved measurement methods of interparticle force between nematic colloids. Although various methods have been utilized for the force measurement, the comparison between the forces obtained by different methods has not been reported. In the frequently used method called the “free-release” method, the hydrodynamic interaction between moving particles has a serious influence on the measurement. In this study we modified those measurement methods by taking the long-ranged hydrodynamic interaction into account. The evaluated forces have been compared with that obtained by the dual beam “optical trap” method, which is free from the hydrodynamic effect. The agreement between them is quantitatively fairly good.


Optical Nonlinearities and Enhanced Light Transmission in Soft-Matter Systems with Tunable Polarizabilities

Weining Man, Shima Fardad, Ze Zhang, Jai Prakash, Michael Lau, Peng Zhang, Matthias Heinrich, Demetrios N. Christodoulides, and Zhigang Chen

We demonstrate a new class of synthetic colloidal suspensions capable of exhibiting negative polarizabilities, and observe for the first time robust propagation and enhanced transmission of self-trapped light over long distances that would have been otherwise impossible in conventional suspensions with positive polarizabilities. Such light penetration through the strong scattering environment is attributed to the interplay between optical forces and self-activated transparency effects while no thermal effect is involved. By judiciously mixing colloidal particles of both negative and positive polarizabilities, we show that the resulting nonlinear response of these systems can be fine-tuned. Our experimental observations are in agreement with theoretical analysis based on a thermodynamic model that takes into account particle-particle interactions. These results may open up new opportunities in developing soft-matter systems with engineered optical nonlinearities.


Bacterial nucleoid structure probed by active drag and resistive pulse sensing

Vivek V. Thacker, Krystyna Bromek, Benoit Meijer, Jurij Kotar, Bianca Sclavi, Marco Cosentino Lagomarsino, Ulrich F. Keyser and Pietro Cicuta

Recent biophysical approaches have provided key insights into the enthalpic and entropic forces that compact the nucleoid in the cell. Our biophysical approach combines two complementary, non-invasive and label-free techniques: a precisely timed steerable optical trap and a high throughput microcapillary Coulter counter. We demonstrate the ability of the latter technique to probe the physical properties and size of many purified nucleoids, at the individual nucleoid level. The DNA-binding protein H-NS is central to the organization of the bacterial genome. Our results show that nucleoids purified from the Δhns strain in the stationary phase expand approximately five fold more than the form observed in WT bacteria. This compaction is consistent with the role played by H-NS in regulating the nucleoid structure and the significant organizational changes that occur as the cell adapts to the stationary phase. We also study the permeability to the flow of ions and find that in the experiment nucleoids behave as solid colloids.


Optical trapping of red blood cells in living animals with a water immersion objective

Min-Cheng Zhong, Lei Gong, Jin-Hua Zhou, Zi-Qiang Wang, and Yin-Mei Li

We demonstrate optical trapping of red blood cells (RBCs) in living animals by using a water immersion objective. First, the cells within biological tissue are mimicked by the particles immersed in aqueous solutions of glycerol. The optical forces depending on trapping depth are investigated when a parallel laser beam enters the water immersion objective. The results show that the optical forces vary with trapping depth, and the optimal trapping depth in aqueous solutions of glycerol (n=1.39) is 50 μm. Second, the optimal trapping depth in aqueous solutions of glycerol can be changed by altering the actual tube length of the water immersion objective. Finally, we achieved optical trapping and manipulation of RBCs in living mice.


Monday, December 9, 2013

Viscoelasticity as a Biomarker for High-Throughput Flow Cytometry

Tobias Sawetzki, Charles D. Eggleton, Sanjay A. Desai, David W.M. Marr

The mechanical properties of living cells are a label-free biophysical marker of cell viability and health; however, their use has been greatly limited by low measurement throughput. Although examining individual cells at high rates is now commonplace with fluorescence activated cell sorters, development of comparable techniques that nondestructively probe cell mechanics remains challenging. A fundamental hurdle is the signal response time. Where light scattering and fluorescence signatures are virtually instantaneous, the cell stress relaxation, typically occurring on the order of seconds, limits the potential speed of elastic property measurement. To overcome this intrinsic barrier to rapid analysis, we show here that cell viscoelastic properties measured at frequencies far higher than those associated with cell relaxation can be used as a means of identifying significant differences in cell phenotype. In these studies, we explore changes in erythrocyte mechanical properties caused by infection with Plasmodium falciparum and find that the elastic response alone fails to detect malaria at high frequencies. At timescales associated with rapid assays, however, we observe that the inelastic response shows significant changes and can be used as a reliable indicator of infection, establishing the dynamic viscoelasticity as a basis for nondestructive mechanical analogs of current high-throughput cell classification methods.


Direct Observation of Phosphate Inhibiting the Force-Generating Capacity of a Miniensemble of Myosin Molecules

Edward P. Debold, Sam Walcott, Mike Woodward, Matthew A. Turner

Elevated levels of phosphate (Pi) reduce isometric force, providing support for the notion that the release of Pi from myosin is closely associated with the generation of muscular force. Pi is thought to rebind to actomyosin in an ADP-bound state and reverse the force-generating steps, including the rotation of the lever arm (i.e., the powerstroke). Despite extensive study, this mechanism remains controversial, in part because it fails to explain the effects of Pi on isometric ATPase and unloaded shortening velocity. To gain new insight into this process, we determined the effect of Pi on the force-generating capacity of a small ensemble of myosin (∼12 myosin heads) using a three-bead laser trap assay. In the absence of Pi, myosin pulled the actin filament out of the laser trap an average distance of 54 ± 4 nm, translating into an average peak force of 1.2 pN. By contrast, in the presence of 30 mM Pi, myosin generated only enough force to displace the actin filament by 13 ± 1 nm, generating just 0.2 pN of force. The elevated Pi also caused a >65% reduction in binding-event lifetime, suggesting that Pi induces premature detachment from a strongly bound state. Definitive evidence of a Pi-induced powerstroke reversal was not observed, therefore we determined if a branched kinetic model in which Pi induces detachment from a strongly bound, postpowerstroke state could explain these observations. The model was able to accurately reproduce not only the data presented here, but also the effects of Pi on both isometric ATPase in muscle fibers and actin filament velocity in a motility assay. The ability of the model to capture the findings presented here as well as previous findings suggests that Pi-induced inhibition of force may proceed along a kinetic pathway different from that of force generation.


Mode-based microparticle conveyor belt in air-filled hollow-core photonic crystal fiber

Oliver A. Schmidt, Tijmen G. Euser, and Philip St.J. Russell

We show how microparticles can be moved over long distances and precisely positioned in a low-loss air-filled hollow-core photonic crystal fiber using a coherent superposition of two co-propagating spatial modes, balanced by a backward-propagating fundamental mode. This creates a series of trapping positions spaced by half the beat-length between the forward-propagating modes (typically a fraction of a millimeter). The system allows a trapped microparticle to be moved along the fiber by continuously tuning the relative phase between the two forward-propagating modes. This mode-based optical conveyor belt combines long-range transport of microparticles with a positional accuracy of 1 µm. The technique also has potential uses in waveguide-based optofluidic systems.


An Optical Trap Combined with Three-Color FRET

Sanghwa Lee and Sungchul Hohng

We developed a hybrid technique combining optical tweezers and single-molecule three-color fluorescence resonance energy transfer (FRET). In demonstrative experiments, we observed the force-sensitive correlated motion of three helical arms of a Holliday junction and identified the independent unfolding/folding dynamics of two DNA hairpins of the same length. With 3 times the number of observable elements of single-molecule FRET, this new instrument will enable the measurement of the complex, multidimensional effects of mechanical forces in various biomolecular systems, such as RNA and proteins.


Mechanosensitive channels and bacterial cell wall integrity: does life end with a bang or a whimper?

Marcel Reuter, Nicholas J. Hayward, Susan S. Black, Samantha Miller, David T. F. Dryden and Ian R. Booth

Mechanogated channels are fundamental components of bacterial cells that enable retention of physical integrity during extreme increases in cell turgor. Optical tweezers combined with microfluidics have been used to study the fate of individual Escherichia coli cells lacking such channels when subjected to a bursting stress caused by increased turgor. Fluorescence-activated cell sorting and electron microscopy complement these studies. These analyses show that lysis occurs with a high probability, but the precise path differs between individual cells. By monitoring the loss of cytoplasmic green fluorescent protein, we have determined that some cells release this protein but remain phase dark (granular) consistent with the retention of the majority of large proteins. By contrast, most cells suffer cataclysmic wall failure leading to loss of granularity but with the retention of DNA and overall cell shape (protein-depleted ghosts). The time span of these events induced by hypo-osmotic shock varies but is of the order of milliseconds. The data are interpreted in terms of the timing of mechanosensitive channel gating relative to osmotically induced water influx.


Optical tweezers technique and its applications

HongLian Guo, ZhiYuan Li

Since their advent in the 1980s, optical tweezers have attracted more and more attention due to their unique non-contact and non-invasion characteristics and their wide applications in physics, biology, chemistry, medical science and nanoscience. In this paper, we introduce the basic principle, the history and typical applications of optical tweezers and review our recent experimental works on the development and application of optical tweezers technique. We will discuss in detail several technological issues, including high precision displacement and force measurement in single-trap and dual-trap optical tweezers, multi-trap optical tweezers with each trap independently and freely controlled by means of space light modulator, and incorporation of cylindrical vector optical beams to build diversified optical tweezers beyond the conventional Gaussian-beam optical tweezers. We will address the application of these optical tweezers techniques to study biophysical problems such as mechanical deformation of cell membrane and binding energy between plant microtubule and microtubule associated proteins. Finally we present application of the optical tweezers technique for trapping, transporting, and patterning of metallic nanoparticles, which can be harnessed to manipulate surface plasmon resonance properties of these nanoparticles.


Crystal Growth of Lysozyme Controlled by Laser Trapping

Jing-Ru Tu, Atsushi Miura, Ken-ichi Yuyama, Hiroshi Masuhara, and Teruki Sugiyama

We demonstrate the growth of a tetragonal crystal of hen egg-white lysozyme (HEWL) in D2O buffer solution controlled by laser trapping with a focused continuous-wave (CW) near-infrared (NIR) laser beam. The focal spot was located at 10 μm away from the edge of the target crystal that was generated spontaneously, and the crystal growth was observed although the focal spot size was much smaller than the distance. The growth rate of (101) and {110} faces of the tetragonal crystal was examined with various laser powers and polarizations. The rate observed under the irradiation was much different from those in spontaneous growth, namely, the growth rate of the {110} face showed a large decrease or increase depending on the irradiation time. The dynamics and mechanism of this unusual crystal growth behavior is discussed from the viewpoint of a large stable domain formation of the HEWL liquidlike clusters through liquid nucleation and growth and by considering the anisotropy of the cluster domain.