Tuesday, May 30, 2017

Resonance Raman and UV-Visible Microscopy Reveals that Conditioning Red Blood Cells with Repeated Doses of Sodium Dithionite Increases Haemoglobin Oxygen Uptake

Dr. David Perez–Guaita, Dr. Marleen de Veij, Dr. Katarzyna M. Marzec, Dr. Abdullah R. D. Almohammedi, Prof. Don McNaughton, Dr. Andrew J. Hudson, Prof. Bayden R. Wood

Here we report that successive additions of fresh dithionite to a suspension of red blood cells (RBCs) increase the capacity of the cells to uptake oxygen. This effect was not observed when the RBCs were similarly preconditioned using gaseous N2 to induce short episodes of hypoxia. The effect of successive sodium dithionite and N2 gas additions on a population of functional erythrocytes was monitored using Raman confocal microscopy, with 514 nm excitation, and UV–visible microscopy. The results indicate that successive additions of sodium dithionite in a suspension of red blood cells leads to an increase in both the rate and the capacity of the RBCs to uptake oxygen. The sodium dithionite did not cause haemoglobin from lysed RBCs to uptake more oxygen after successive additions and hence this effect was only observed in functional intact RBCs. Experiments performed with polarised Raman spectroscopy suggest that sodium dithionite increases the disorder of Hb in the RBC facilitating oxygen diffusion.


Evanescent field interferometric optical tweezers with rotational symmetric patterns

Mohammadbagher Mohammadnezhad and Abdollah Hassanzadeh

A simple configuration to create optical lattices with 𝑁N-fold rotational symmetry using the interference of 𝑁N evanescent fields is proposed. The potential energy of a Rayleigh particle in the generated patterns (optical lattices) is investigated. The results show that by adjusting the relative phases and polarization states of the incident beams, an enormous range of optical lattices can be obtained. When the number of interfering beams is increased, circular spots beyond the diffraction limit can be created. For 𝑠s-polarization, the intensity landscape has a dark-centered distribution at the origin encircled by a very small bright circular ring whose radius is 120 nm. This intensity distribution acts as a hollow beam and can be used for efficient trapping of low-index particles. We believe that the resulting lattices can find applications in the arrangement of particles, creation of optical lattices, and efficient trapping of very small Rayleigh particles.


Light-driven crystallization of polystyrene micro-spheres

Jing Liu and Zhi-Yuan Li

We investigate the dynamic crystallization processes of colloidal photonic crystals, which are potentially invaluable for solving a number of existing and emerging technical problems in regards to controlled fabrication of crystals, such as size normalization, stability improvement, and acceleration of synthesis. In this paper, we report systematic high-resolution optical observation of the spontaneous crystallization of monodisperse polystyrene (PS) micro-spheres in aqueous solution into close-packed arrays in a static line optical tweezers. The experiments demonstrate that the crystal structure is mainly affected by the minimum potential energy of the system; however, the crystallization dynamics could be affected by various mechanical, physical, and geometric factors. The complicated dynamic transformation process from 1D crystallization to 2D crystallization and the creation and annihilation of dislocations and defects via crystal relaxation are clearly illustrated. Two major crystal growth modes, the epitaxy growth pattern and the inserted growth pattern, have been identified to play a key role in shaping the dynamics of the 1D and 2D crystallization process. These observations offer invaluable insights for in-depth research about colloidal crystal crystallization.


Interactions between the breast cancer-associated MUC1 mucins and C-type lectin characterized by optical tweezers

Soosan Hadjialirezaei, Gianfranco Picco, Richard Beatson, Joy Burchell, Bjørn Torger Stokke, Marit Sletmoen

Carbohydrate–protein interactions govern many crucial processes in biological systems including cell recognition events. We have used the sensitive force probe optical tweezers to quantify the interactions occurring between MGL lectins and MUC1 carrying the cancer-associated glycan antigens mucins Tn and STn. Unbinding forces of 7.6 pN and 7.1 pN were determined for the MUC1(Tn)—MGL and MUC1(STn)—MGL interactions, at a force loading rate of ~40 pN/s. The interaction strength increased with increasing force loading rate, to 27 and 37 pN at a force loading rate of ~ 310 pN/s. No interactions were detected between MGL and MUC1(ST), a glycoform of MUC1 also expressed by breast carcinoma cells. Interestingly, this glycan (ST) can be found on proteins expressed by normal cells, although in this case not on MUC1. Additionally, GalNAc decorated polyethylene glycol displayed similar rupture forces as observed for MUC1(Tn) and MUC1(STn) when forced to unbind from MGL, indicating that GalNAc is an essential group in these interactions. Since the STn glycan decoration is more frequently found on the surface of carcinomas than the Tn glycan, the binding of MUC1 carrying STn to MGL may be more physiologically relevant and may be in part responsible for some of the characteristics of STn expressing tumours.


Laser Capture Microdissection of Pancreatic Acinar Cells to Identify Proteomic Alterations in a Murine Model of Caerulein-Induced Pancreatitis

John P Shapiro, Hannah M Komar, Baris Hancioglu, Lianbo Yu, Ming Jin, Yuko Ogata, Phil A Hart, Zobeida Cruz-Monserrate, Gregory B Lesinski and Darwin L Conwell

Chronic pancreatitis (CP) is characterized by inflammation and fibrosis of the pancreas, leading to pain, parenchymal damage, and loss of exocrine and endocrine function. There are currently no curative therapies; diagnosis remains difficult and aspects of pathogenesis remain unclear. Thus, there is a need to identify novel biomarkers to improve diagnosis and understand pathophysiology. We hypothesize that pancreatic acinar regions contain proteomic signatures relevant to disease processes, including secreted proteins that could be detected in biofluids. Acini from pancreata of mice injected with or without caerulein were collected using laser capture microdissection followed by mass spectrometry analysis. This protocol enabled high-throughput analysis that captured altered protein expression throughout the stages of CP. Over 2,900 proteins were identified, whereas 331 were significantly changed ≥2-fold by mass spectrometry spectral count analysis. Consistent with pathogenesis, we observed increases in proteins related to fibrosis (e.g., collagen, P<0.001), several proteases (e.g., trypsin 1, P<0.001), and altered expression of proteins associated with diminished pancreas function (e.g., lipase, amylase, P<0.05). In comparison with proteomic data from a public data set of CP patients, a significant correlation was observed between proteomic changes in tissue from both the caerulein model and CP patients (r=0.725, P<0.001). This study illustrates the ability to characterize proteome changes of acinar cells isolated from pancreata of caerulein-treated mice and demonstrates a relationship between signatures from murine and human CP.


Plasmonic Trapping and Release of Nanoparticles in a Monitoring Environment

Jung-Dae Kim, Yong-Gu Lee

Plasmonic tweezers use surface plasmon polaritons to confine polarizable nanoscale objects. Among the various designs of plasmonic tweezers, only a few can observe immobilized particles. Moreover, a limited number of studies have experimentally measured the exertable forces on the particles. The designs can be classified as the protruding nanodisk type or the suppressed nanohole type. For the latter, microscopic observation is extremely challenging. In this paper, a new plasmonic tweezer system is introduced to monitor particles, both in directions parallel and orthogonal to the symmetric axis of a plasmonic nanohole structure. This feature enables us to observe the movement of each particle near the rim of the nanohole. Furthermore, we can quantitatively estimate the maximal trapping forces using a new fluidic channel.


Friday, May 26, 2017

Effects of photon scattering torque in off-axis levitated torsional cavity optomechanics

M. Bhattacharya, B. Rodenburg, W. Wetzel, B. Ek, and A. K. Jha

We consider theoretically a dielectric nanoparticle levitated in an optical ring trap inside a cavity and probed by an angular lattice, with all electromagnetic fields carrying orbital angular momentum. Analyzing the torsional motion of the particle about the cavity axis, we find that photon scattering from the trap beam plays an important role in the optomechanical system. First we show that the presence of the torque introduces an instability. Subsequently, we demonstrate that for bound motion near a stable equilibrium, varying the optical torque strength allows for tuning the linear optomechanical coupling. Finally, we indicate that the relative strengths of the linear and quadratic couplings can be detected directly by homodyning the cavity output. Our studies should be of interest to researchers exploring quantum mechanics using torsional optomechanics.


Measurement of mass by optical forced oscillation of absorbing particles trapped in air

Jinda Lin, Jianliao Deng, Rong Wei, Yong-qing Li, and Yuzhu Wang

We demonstrate the application of optical forced oscillation to measure the mass of an absorbing microparticle trapped in air. When the light intensity is modulated sinusoidally, the particle in the trap undergoes forced oscillation, and the amplitude of the oscillation depends directly on the modulation frequency. We obtain the stiffness of the optical trap and the mass of the trapped particle by fitting the experimental data of the amplitudes versus the modulation frequencies with a simple spring model. The fitting results show that, for a certain particle, the stiffness varies linearly with the trapping light intensity while the mass is constant. The density of the microparticle is also estimated, which has potential to classify different kinds of absorbing particles, such as C and CuO.


Levitated nanoparticle as a classical two-level atom

Martin Frimmer, Jan Gieseler, Thomas Ihn, and Lukas Novotny

The center-of-mass motion of a single optically levitated nanoparticle resembles three uncoupled harmonic oscillators. We show how a suitable modulation of the optical trapping potential can give rise to a coupling between two of these oscillators, such that their dynamics are governed by a classical equation of motion that resembles the Schrödinger equation for a two-level system. Based on experimental data, we illustrate the dynamics of this parametrically coupled system both in the frequency and in the time domain. We discuss the limitations and differences of the mechanical analog in comparison to a true quantum-mechanical system.


Exogenous lysophospholipids with large head groups perturb clathrin-mediated endocytosis

Ieva Ailte, Anne Berit D. Lingelem, Audun S. Kvalvaag, Simona Kavaliauskiene, Andreas Brech, Gerbrand Koster, Paul G. Dommersnes, Jonas Bergan, Tore Skotland, Kirsten Sandvig

In this study, we have investigated how clathrin-dependent endocytosis is affected by exogenously added lysophospholipids (LPLs). Addition of LPLs with large head groups strongly inhibits transferrin (Tf) endocytosis in various cell lines, while LPLs with small head groups do not. Electron and total internal reflection fluorescence microscopy (EM and TIRF) reveal that treatment with lysophosphatidylinositol (LPI) with the fatty acyl group C18:0 leads to reduced numbers of invaginated clathrin-coated pits (CCPs) at the plasma membrane, fewer endocytic events per membrane area and increased lifetime of CCPs. Also, endocytosis of Tf becomes dependent on actin upon LPI treatment. Thus, our results demonstrate that one can regulate the kinetics and properties of clathrin-dependent endocytosis by addition of LPLs in a head group size- and fatty acyl-dependent manner. Furthermore, studies performed with optical tweezers show that less force is required to pull membrane tubules outwards from the plasma membrane when LPI is added to the cells. The results are in agreement with the notion that insertion of LPLs with large head groups creates a positive membrane curvature which might have a negative impact on events that require plasma membrane invagination, while it may facilitate membrane bending toward the cell exterior.

Characterization of facilitated diffusion of tumor suppressor p53 along DNA using single-molecule fluorescence imaging

Kiyoto Kamagata, Agato Murata, Yuji Itoh, Satoshi Takahashi

Sequence-specific DNA-binding proteins can maintain and regulate cellular functions by accurately and quickly binding to target sequences among large amounts of nontarget DNA. The facilitated diffusion mechanism of DNA-binding proteins—a combination of three-dimensional (3D) diffusion and one-dimensional (1D) sliding along DNA—has been proposed to explain the target binding accuracy and rapidity and has been partially confirmed experimentally. Nonetheless, quantitative elucidation of the mechanism has remained difficult. Furthermore, many additional steps in facilitated diffusion have been proposed. In this review, we introduce the theoretical and experimental studies and the current understanding of facilitated diffusion of DNA-binding proteins. We focused on tumor suppressor p53 as a key protein subject to facilitated diffusion; p53 regulates various cellular processes such as cell cycle arrest, DNA repair, and apoptosis upon binding to a target sequence of DNA after activation by external stress to the cell. We describe the research on the 3D diffusion and 1D sliding of p53 mainly via single-molecule fluorescence microscopy. In addition to the demonstration of the 1D sliding of p53, recent experiments revealed multiple modes of 1D sliding, regulation of the target recognition, and the constant search distance despite changes in the concentrations of divalent cations. Furthermore, rotation-coupled 1D sliding along DNA is suggested. A comparison of parameters of the facilitated diffusion of p53 and those of other DNA-binding proteins characterized so far suggests that the ratio of 3D diffusion and 1D sliding is close to the theoretical optimum of 1:1 for several proteins including p53.


Thursday, May 25, 2017

Effects of Relative Humidity and Particle Phase Water on the Heterogeneous OH Oxidation of 2-Methylglutaric Acid Aqueous Droplets

Man Mei Chim, Chun Yin Chow, James F. Davies, and Man Nin Chan

Organic aerosols can exist as aqueous droplets, with variable water content depending on their composition and environmental conditions (e.g., relative humidity (RH)). Recent laboratory studies have revealed that oxidation kinetics in highly concentrated droplets can be much slower than those in dilute solutions. However, it remains unclear whether aerosol phase water affects the formation of reaction products physically and/or chemically. In this work, we investigate the role of aerosol phase water on the heterogeneous chemistry of aqueous organic droplets consisting of 2-methylglutaric acid (2-MGA), measuring the reaction kinetics and the reaction products upon heterogeneous OH oxidation over a range of RH. An atmospheric pressure soft ionization source (direct analysis in real time, DART) coupled with a high-resolution mass spectrometer is used to obtain real-time molecular information on the reaction products. Aerosol mass spectra show that the same reaction products are formed at all measured RH. At a given reaction extent of the parent 2-MGA, the aerosol composition is independent of RH. These results suggest the aerosol phase water does not alter reaction mechanisms significantly. Kinetic measurements find that the effective OH uptake coefficient, γeff, decreases with decreasing RH below 72%. Isotopic exchange measurements performed using aerosol optical tweezers reveal water diffusion coefficients in the 2-MGA droplets to be 3.0 × 10–13 to 8.0 × 10–13 m2 s–1 over the RH range of 47–58%. These values are comparable to those of other viscous organic aerosols (e.g., citric acid), indicating that 2-MGA droplets are likely to be viscous at low humidity. Smaller γeff at low RH is likely attributed to the slower diffusion of reactants within the droplets. Taken together, the observed relationship between the γeff and RH is likely attributed to changes in aerosol viscosity rather than changes in reaction mechanisms.


Human RAD52 Captures and Holds DNA Strands, Increases DNA Flexibility, and Prevents Melting of Duplex DNA: Implications for DNA Recombination

Ineke Brouwer, Hongshan Zhang, Andrea Candelli, Davide Normanno, Erwin J.G. Peterman, Gijs J.L. Wuite, Mauro Modesti

Human RAD52 promotes annealing of complementary single-stranded DNA (ssDNA). In-depth knowledge of RAD52-DNA interaction is required to understand how its activity is integrated in DNA repair processes. Here, we visualize individual fluorescent RAD52 complexes interacting with single DNA molecules. The interaction with ssDNA is rapid, static, and tight, where ssDNA appears to wrap around RAD52 complexes that promote intra-molecular bridging. With double-stranded DNA (dsDNA), interaction is slower, weaker, and often diffusive. Interestingly, force spectroscopy experiments show that RAD52 alters the mechanics dsDNA by enhancing DNA flexibility and increasing DNA contour length, suggesting intercalation. RAD52 binding changes the nature of the overstretching transition of dsDNA and prevents DNA melting, which is advantageous for strand clamping during or after annealing. DNA-bound RAD52 is efficient at capturing ssDNA in trans. Together, these effects may help key steps in DNA repair, such as second-end capture during homologous recombination or strand annealing during RAD51-independent recombination reactions.


Characterization of biomechanical properties of cells through dielectrophoresis-based cell stretching and actin cytoskeleton modeling

Guohua Bai, Ying Li, Henry K. Chu, Kaiqun Wang, Qiulin Tan, Jijun Xiong and Dong Sun

Cytoskeleton is a highly dynamic network that helps to maintain the rigidity of a cell, and the mechanical properties of a cell are closely related to many cellular functions. This paper presents a new method to probe and characterize cell mechanical properties through dielectrophoresis (DEP)-based cell stretching manipulation and actin cytoskeleton modeling. Leukemia NB4 cells were used as cell line, and changes in their biological properties were examined after chemotherapy treatment with doxorubicin (DOX). DEP-integrated microfluidic chip was utilized as a low-cost and efficient tool to study the deformability of cells. DEP forces used in cell stretching were first evaluated through computer simulation, and the results were compared with modeling equations and with the results of optical stretching (OT) experiments. Structural parameters were then extracted by fitting the experimental data into the actin cytoskeleton model, and the underlying mechanical properties of the cells were subsequently characterized. The DEP forces generated under different voltage inputs were calculated and the results from different approaches demonstrate good approximations to the force estimation. Both DEP and OT stretching experiments confirmed that DOX-treated NB4 cells were stiffer than the untreated cells. The structural parameters extracted from the model and the confocal images indicated significant change in actin network after DOX treatment. The proposed DEP method combined with actin cytoskeleton modeling is a simple engineering tool to characterize the mechanical properties of cells.


Targeting Tumor-Associated Exosomes with Integrin-Binding Peptides

Randy P. Carney, Sidhartha Hazari, Tatu Rojalin, Alisha Knudson, Tingjuan Gao, Yuchen Tang, Ruiwu Liu, Tapani Viitala, Marjo Yliperttula, Kit S. Lam

All cells expel a variety of nanosized extracellular vesicles (EVs), including exosomes, with composition reflecting the cells' biological state. Cancer pathology is dramatically mediated by EV trafficking via key proteins, lipids, metabolites, and microRNAs. Recent proteomics evidence suggests that tumor-associated exosomes exhibit distinct expression of certain membrane proteins, rendering those proteins as attractive targets for diagnostic or therapeutic application, yet it is not currently feasible to distinguish circulating EVs in complex biofluids according to their tissue of origin or state of disease. Here, peptide binding to tumor-associated EVs via overexpressed membrane protein is demonstrated. It is found that SKOV-3 ovarian tumor cells and their released EVs express α3β1 integrin, which can be targeted by the in-house cyclic nonapeptide, LXY30. After measuring bulk SKOV-3 EV association with LXY30 by flow cytometry, Raman spectral analysis of laser-trapped single exosomes with LXY30-dialkyne conjugate enables the differentiation of cancer-associated exosomes from noncancer exosomes. Furthermore, the foundation for a highly specific detection platform for tumor-EVs in solution with biosensor surface-immobilized LXY30 is introduced. LXY30 not only exhibits high specificity and affinity to α3β1 integrin-expressing EVs, but also reduces EV uptake into SKOV-3 parent cells, demonstrating the possibility for therapeutic application.


Optimizing phase to enhance optical trap stiffness

Michael A. Taylor

Phase optimization offers promising capabilities in optical tweezers, allowing huge increases in the applied forces, trap stiff-ness, or measurement sensitivity. One key obstacle to potential applications is the lack of an efficient algorithm to compute an optimized phase profile, with enhanced trapping experiments relying on slow programs that would take up to a week to converge. Here we introduce an algorithm that reduces the wait from days to minutes. We characterize the achievable in-crease in trap stiffness and its dependence on particle size, refractive index, and optical polarization. We further show that phase-only control can achieve almost all of the enhancement possible with full wavefront shaping; for instance phase control allows 62 times higher trap stiffness for 10 μm silica spheres in water, while amplitude control and non-trivial polarization further increase this by 1.26 and 1.01 respectively. This algorithm will facilitate future applications in optical trapping, and more generally in wavefront optimization.


All-Optical Chirality-Sensitive Sorting via Reversible Lateral Forces in Interference Fields

Tianhang Zhang, Mahdy Rahman Chowdhury Mahdy, Yongmin Liu, Jing Hua Teng, Chwee Teck Lim, Zheng Wang, and Cheng-Wei Qiu

Separating substances by their chirality faces great challenges as well as opportunities in chemistry and biology. In this study, we propose an all-optical solution for passive sorting of chiral objects using chirality-dependent lateral optical forces induced by judiciously interfered fields. First, we investigate the optical forces when the chiral objects are situated in the interference field formed by two plane waves with arbitrary polarization states. When the plane waves are either linearly or circularly polarized, nonzero lateral forces are found at the particle’s trapping positions, making such sideways motions observable. Although the lateral forces have different magnitudes on particles with different chirality, their directions are the same for opposite handedness particles, rendering it difficult to separate the chiral particles. We further solve the sorting problem by investigating more complicated polarization states. Finally, we achieve the chiral-selective separation by illuminating only one beam toward the chiral substance situated at an interface between two media, taking advantage of the native interference between the incident and reflective beams at the interface. Our study provides a robust and insightful approach to sort chiral substances and biomolecules with plausible optical setups.


Wednesday, May 24, 2017

Carbonylation of atrial myosin prolongs its interaction with actin

G. Kopylova, S. Nabiev, D. Shchepkin, S. Bershitsky

Carbonylation induced by hyperthyroidism suppresses force generation of skeletal myosin and sliding velocity of actin filaments in an in vitro motility assay. However, its effects on cardiac myosin at the molecular level have not been studied. Hyperthyroidism induces a change in expression of myosin heavy chains in ventricles, which may mask the effect of oxidation. In contrast to ventricular myosin, expression of myosin heavy chains in the atrium does not change upon hyperthyroidism and enables investigation of the effect of oxidation on cardiac myosin. We studied the influence of carbonylation, a type of protein oxidation, on the motor function of atrial myosin and Ca2+ regulation of actin-myosin interaction at the level of isolated proteins and single molecules using an in vitro motility assay and an optical trap. Carbonylation of atrial myosin prolonged its attached state on actin and decreased maximal sliding velocity of thin filaments over this myosin but did not affect the calcium sensitivity of the velocity. The results indicate that carbonylation of atrial myosin induced by hyperthyroidism can be a rate-limiting factor of atrium contractility and so participates in the genesis of heart failure in hyperthyroidism.


Observation of spin and orbital rotation of red blood cell in dual-beam fibre-optic trap with transverse offset

Xinlin Chen, Guangzong Xiao, Xiang Han, Wei Xiong, Hui Luo and Baoli Yao

The spin and orbital rotation of the red blood cell (RBC) are achieved simultaneously by introducing a transverse offset to the dual-beam fibre-optic trap. The motion type of the captured RBC could be controlled by adjusting the offset distance. When the offset distance is relatively small, the RBC is observed to spin in the trap centre, with the spin frequency increasing linearly with the offset distance. Once the offset distance is above a critical value, the RBC will rotate along an elliptic orbit, together with the spin motion. The orbital rotation frequency and spin frequency both decrease with the increased offset distance. This technique allows mixing and viewing living cells from different perspectives concurrently without exposing them to any mechanical contact, and is generally applicable to biological and medical research.


Luminescence Dynamics of Silica-Encapsulated Quantum Dots During Optical Trapping

Héctor Rodríguez-Rodríguez, María Acebrón, Beatriz H. Juárez, and J. Ricardo Arias-Gonzalez

The trade-off between photobrightening and photobleaching controls the emission stability of colloidal quantum dots. This balance is critical in optical trapping configurations, where irradiances that confine and simultaneously excite the nanocrystals in the focal region cannot be indefinitely lowered. In this work, we studied the photobrightening and bleaching behaviors of two types of silica-encapsulated quantum dots excited upon two-photon absorption in an optical trap. The first type consists of alloyed CdSeZnS quantum dots covered with a silica shell. We found that the dynamics of these as-prepared architectures are similar to those previously reported for bare surface-deposited quantum dots, where thousands of times smaller irradiances were used. We then analyzed the same quantum dot systems treated with an extra intermediate sulfur passivating shell for the better understanding of the surface traps influence in the temporal evolution of their emission in the optical trap. We found that these latter systems exhibit better homogeneity in their photodynamic behavior compared to the untreated ones. These features strengthen the value of quantum dot preparations in optical manipulation as well as for applications where both long and maximal emission stability in physiological and other polar media are required.


Back-focal-plane displacement detection using side-scattered light in dual-beam fiber-optic traps

Wei Xiong, Guangzong Xiao, Xiang Han, Jinhua Zhou, Xinlin Chen, and Hui Luo

In optical traps the position of a trapped bead is usually determined by measuring the intensity distribution of the forward-scattered light and the back-scattered light. In this paper we demonstrate that this position can be determined using the side-scattered light. A quadrant photodiode is used to monitor the position of an optically trapped object in a dual-beam fiber-optic trap by measurement of intensity shifts in the back focal plane of the objective that is perpendicular to the propagating beam. An approximated model based on ray optics is presented with numerical results that describe the use of the side-scattered light for position detection. The influences of system parameters, including fiber separations, the numerical apertures (NA), and the radii of microspheres, are discussed in details. We find out that the displacement sensitivity of the detector is null for some critical radii and numerical apertures. In addition, the noises in laser powers are analyzed, and one power difference regime is proposed to weaken the influences.


Tuesday, May 23, 2017

Measurement of the Raman spectra and hygroscopicity of four pharmaceutical aerosols as they travel from pressurised metered dose inhalers (pMDI) to a model lung

N. Davidson, H.-J. Tong, M. Kalberer, P.C. Seville, A.D. Ward, M.K. Kuimova, F.D. Pope

Particle inhalation is an effective and rapid delivery method for a variety of pharmaceuticals, particularly bronchodilation drugs used for treating asthma and COPD. Conditions of relative humidity and temperature inside the lungs are generally very different from the outside ambient air, with the lung typically being warmer and more humid. Changes in humidity, from inhaler to lung, can cause hygroscopic phase transitions and particle growth. Increasing particle size and mass can negatively affect particle deposition within the lung leading to inefficient treatment, while deliquescence prior to impaction is liable to accelerate drug uptake. To better understand the hygroscopic properties of four pharmaceutical aerosol particles; pharmaceutical particles from four commercially available pressurised metered dose inhalers (pMDIs) were stably captured in an optical trap, and their composition was examined online via Raman spectroscopy. Micron-sized particles of salbutamol sulfate, salmeterol xinafoate, fluticasone propionate and ciclesonide were levitated and examined over a range of relative humidity values inside a chamber designed to mimic conditions within the respiratory tract. The effect of temperature upon hygroscopicity was also investigated for salbutamol sulfate particles. Salbutamol sulfate was found to have significant hygroscopicity, salmeterol xinafoate showed some hygroscopic interactions, whilst fluticasone propionate and ciclesonide revealed no observable hygroscopicity. Thermodynamic and structural modelling is used to explain the observed experimental results.


A systematic experimental study on the evaporation rate of supercooled water droplets at subzero temperatures and varying relative humidity

S. Ruberto, J. Reutzsch, N. Roth, B. Weigand

Supercooled water droplets (SWD) are present in clouds at high altitude and subjected to very low temperatures and high relative humidity. These droplets exist in a metastable state. The understanding of the evaporation of SWD at these extreme conditions is of high interest to understand rain, snow, and hail generating mechanisms in clouds. This paper focuses on the experimental results of the measurements of the evaporation rates ββ of supercooled water droplets. For this purpose, single SWDs are trapped by means of optical levitation. During the evaporation process, the elastically scattered light in the forward regime is recorded and evaluated. Experiments have been performed for different relative humidities ϕϕ at three constant ambient temperatures, namely, T∞=268.15; 263.15; 253.15 KT∞=268.15; 263.15; 253.15 K (t∞=−5;−10;−20 ∘Ct∞=−5;−10;−20 ∘C). The experimental data agrees well with direct numerical simulations (DNS) carried out with the in-house code Free Surface 3D (FS3D) and shows that the use of a simplified model is permissible for these ambient conditions.


Probing the germination kinetics of ethanol-treated Bacillus thuringiensis spores

Guiwen Wang, Huanjun Chen, Xiaochun Wang, Lixin Peng, Yuan Peng, and Yong-qing Li

Bacillus thuringiensis (Bt) is the most widely used microbial insecticide. To clarify the mechanism of bacterial resistance to ethanol toxicity, the present study investigated the effects of 70% (v/v) ethanol at a moderate temperature (65°C) on Bt spore germination by single-cell Raman spectroscopy and differential interference contrast microscopy. We found that over 80% of Bt spores were inviable after 30 min of treatment. Moreover, ethanol treatment affected spore germination; the time for initiation of rapid calcium dipicolinate (CaDPA) release (i.e., lag time, 𝑇lagTlag), time taken for rapid CaDPA release (i.e., Δ𝑇releaseΔTrelease), and time required for complete hydrolysis of the peptidoglycan cortex of spores (i.e., Δ𝑇lysΔTlys) were increased with longer treatment times. Alanine-initiated germination upon ethanol treatment for 30–90 min showed a 2- to 4-fold longer 𝑇lagTlag, 2- to 3.5-fold longer Δ𝑇releaseΔTrelease, and ∼2∼2-fold longer Δ𝑇lysΔTlys relative to the control. Dodecylamine-initiated germination treated for 15–30 min had 3- to 5-fold longer 𝑇lagTlag and 1.4- to 1.7-fold longer Δ𝑇releaseΔTrelease than the control. Germination induced by exogenous CaDPA was observed only in a small fraction of spores treated with ethanol for 5 min. Single-cell Raman spectroscopy revealed that more than 52% of spores lost CaDPA after 30 min of ethanol treatment; these showed reductions in the intensity of 1280 and 1652 cm−11652 cm−1 bands (corresponding to protein 𝛼α-helical structure) and increases in that of 1245 and 1665 cm−11665 cm−1 bands (attributed to irregularities in protein structure). These results indicate that CaDPA in the core of Bt spores confers resistance to ethanol, and that damage to the spore inner membrane by ethanol treatment results in CaDPA leakage. Additionally, moderate-temperature ethanol treatment and consequent denaturation of germination-related proteins affected spore germination, specifically by inactivating the cortex-lytic enzyme CwlJ. Our findings provide a theoretical basis for the development of more effective methods for killing spore-forming bacteria; microscopy imaging and Raman spectroscopy can provide novel insight into the effects of chemical agents on microbial cells.


Tunable optical cage array generated by Dammann vector beam

Xiaoyu Weng, Luping Du, Peng Shi, and Xiaocong Yuan

Optical cages attract considerable attentions recently owing to their potential applications in optical trapping, optical imaging and optical cloaking. However, the generation of tunable optical cage arrays in the transverse plane comes to a great challenge, which restricts the effectiveness of the above applications. In this work, we propose a full polarization-controlled method that optical cage arrays with tunable number and positions in the x-y plane can be generated by a so-called Dammann vector beam (DVB), both under the conditions of high- and low-NA focusing system. By adjusting the polarization state of the DVB with the phase of Dammann grating, the number and positions of optical cages can be adjusted flexibly. This work reveals the relationship between the complex polarization state of an incident vector beam and the output optical cage array in the focal region, and may find valuable applications in optical imaging, optical trapping, etc.


Friday, May 19, 2017

Pulling cylindrical particles using a soft-nonparaxial tractor beam

Andrey Novitsky, Weiqiang Ding, Maoyan Wang, Dongliang Gao, Andrei V. Lavrinenko & Cheng-Wei Qiu

In order to pull objects towards the light source a single tractor beam inevitably needs to be strongly nonparaxial. This stringent requirement makes such a tractor beam somewhat hypothetical. Here we reveal that the cylindrical shape of dielectric particles can effectively mitigate the nonparaxiality requirements, reducing the incidence angle of the partial plane waves of the light beam down to 45° and even to 30° for respectively dipole and dipole-quadrupole objects. The optical pulling force attributed to the interaction of magnetic dipole and magnetic quadrupole moments of dielectric cylinders occurs due to the TE rather than TM polarization. Therefore, the polarization state of the incident beam can be utilized as an external control for switching between the pushing and pulling forces. The results have application values towards optical micromanipulation, transportation and sorting of targeted particles.


Chiral nanoparticles in singular light fields

Ilia A. Vovk, Anvar S. Baimuratov, Weiren Zhu, Alexey G. Shalkovskiy, Alexander V. Baranov, Anatoly V. Fedorov & Ivan D. Rukhlenko

The studying of how twisted light interacts with chiral matter on the nanoscale is paramount for tackling the challenging task of optomechanical separation of nanoparticle enantiomers, whose solution can revolutionize the entire pharmaceutical industry. Here we calculate optical forces and torques exerted on chiral nanoparticles by Laguerre–Gaussian beams carrying a topological charge. We show that regardless of the beam polarization, the nanoparticles are exposed to both chiral and achiral forces with nonzero reactive and dissipative components. Longitudinally polarized beams are found to produce chirality densities that can be 109 times higher than those of transversely polarized beams and that are comparable to the chirality densities of beams polarized circularly. Our results and analytical expressions prove useful in designing new strategies for mechanical separation of chiral nanoobjects with the help of highly focussed beams.


Optical Bessel tractor beam on active dielectric Rayleigh prolate and oblate spheroids

F. G. Mitri

Optical Bessel tractor beams, designed to produce a negative pulling force on a particle, are gaining increased attention for applications in noncontact remote sampling, particle manipulation, and handling, to name some examples. In the long-wavelength (Rayleigh) limit, known also as the electric dipole approximation, earlier investigations demonstrated that a zeroth-order Bessel beam incident upon a passive dielectric sphere (i.e., no radiating sources in its core) always acts as a repulsor beam, which causes the particle to be pushed away from the source in the forward direction of the linear momentum. In contrast to what has already been established, this work shows that the incident wave field can act as a tractor beam (where a small spheroid is pulled backwards towards the source due to a negative attractive force) in the dipole approximation (Rayleigh) limit, provided that the particle is made of an active material, i.e., a dielectric spheroid acting as an oscillating source for which the extinction energy efficiency is negative. Numerical computations for the Cartesian components of the optical radiation force on active prolate and oblate spheroids with arbitrary orientation are performed. Emphasis is placed on the emergence of the tractor beam behavior and its dependence upon the half-cone angle, the polarization type of the incident beam, the spheroid aspect ratio, as well as its orientation in space. The analysis is extended to calculate the Cartesian components of the spin radiation torque, which causes a rotation of the spheroid around its center of mass in either the counterclockwise or the clockwise (negative) direction of spinning. Unlike the case of a sphere, the optical spin torque arises for a nonabsorptive oblate or prolate spheroid with arbitrary orientation in the field of a zeroth-order Bessel beam. Potential applications in optically engineered metamaterials, optical tractor beams, tweezers, particle manipulation, rotation, and handling would benefit from the results of this study.


Optical pulling force and torques on Rayleigh semiconductor prolate and oblate spheroids in Bessel tractor beams

F.G. Mitri

Optical tractor Bessel beams are gaining increased interest where a negative attractive force acting in opposite direction of wave propagation is harnessed for particle manipulation in opto-fluidics, the manufacturing of periodic composite metamaterials and other related applications. Previous works considered the spherical geometry, however, it is of some importance to develop improved models to investigate objects of more complex shapes and study the tractor beam effect on them. The aim of this work is therefore directed toward this goal, where the dipole approximation method is used to compute the optical force, spin and orbital torques on a subwavelength semiconductor spheroid illuminated by a zeroth-order Bessel vector beam. Numerical computations for the Cartesian components of the optical radiation force on prolate and oblate spheroids with arbitrary orientation are performed, with emphasis on the emergence of a negative pulling force and its dependence on the half-cone angle of the beam, the aspect ratio of the spheroid, and its orientation in space. Moreover, the Cartesian components of the spin radiation torque are computed where a negative spin torque can arise, which causes a rotational twisting effect of the spheroid around its center of mass in either the counterclockwise or the clockwise (negative) direction of spinning. In addition, the axial component of the orbital radiation torque is computed which also shows sign reversal. The results of this analysis provide a priori information for the design and development of novel optical tweezers devices and tractor beams, with potential applications in the manipulation and handling of elongated particles.


Brownian motion as a new probe of wettability

Jianyong Mo, Akarsh Simha, and Mark G. Raizen

Understanding wettability is crucial for optimizing oil recovery, semiconductor manufacturing, pharmaceutical industry, and electrowetting. In this letter, we study the effects of wettability on Brownian motion. We consider the cases of a sphere in an unbounded fluid medium, as well as a sphere placed in the vicinity of a plane wall. For the first case, we show the effects of wettability on the statistical properties of the particles’ motion, such as velocity autocorrelation, velocity, and thermal force power spectra over a large range of time scales. We also propose a new method to measure wettability based on the particles’ Brownian motion. In addition, we compare the boundary effects on Brownian motion imposed by both no-slip and perfect-slip flat walls. We emphasize the surprising boundary effects on Brownian motion imposed by a perfect-slip wall in the parallel direction, such as a higher particle mobility parallel to a perfect flat wall compared to that in the absence of the wall, as well as compared to a particle near a no-slip flat wall.


Wednesday, May 17, 2017

Application of Microrheology in Food Science

Nan Yang, Ruihe Lv, Junji Jia, Katsuyoshi Nishinari, and Yapeng Fang

Microrheology provides a technique to probe the local viscoelastic properties and dynamics of soft materials at the microscopic level by observing the motion of tracer particles embedded within them. It is divided into passive and active microrheology according to the force exerted on the embedded particles. Particles are driven by thermal fluctuations in passive microrheology, and the linear viscoelasticity of samples can be obtained on the basis of the generalized Stokes-Einstein equation. In active microrheology, tracer particles are controlled by external forces, and measurements can be extended to the nonlinear regime. Microrheology techniques have many advantages such as the need for only small sample amounts and a wider measurable frequency range. In particular, microrheology is able to examine the spatial heterogeneity of samples at the microlevel, which is not possible using traditional rheology. Therefore, microrheology has considerable potential for studying the local mechanical properties and dynamics of soft matter, particularly complex fluids, including solutions, dispersions, and other colloidal systems. Food products such as emulsions, foams, or gels are complex fluids with multiple ingredients and phases. Their macroscopic properties, such as stability and texture, are closely related to the structure and mechanical properties at the microlevel. In this article, the basic principles and methods of microrheology are reviewed, and the latest developments and achievements of microrheology in the field of food science are presented.


Elasticity of the transition state for oligonucleotide hybridization

Kevin D. Whitley Matthew J. Comstock Yann R. Chemla

Despite its fundamental importance in cellular processes and abundant use in biotechnology, we lack a detailed understanding of the kinetics of nucleic acid hybridization. In particular, the identity of the transition state, which determines the kinetics of the two-state reaction, remains poorly characterized. Here, we used optical tweezers with single-molecule fluorescence to observe directly the binding and unbinding of short oligonucleotides (7–12 nt) to a complementary strand held under constant force. Binding and unbinding rate constants measured across a wide range of forces (1.5–20 pN) deviate from the exponential force dependence expected from Bell's equation. Using a generalized force dependence model, we determined the elastic behavior of the transition state, which we find to be similar to that of the pure single-stranded state. Our results indicate that the transition state for hybridization is visited before the strands form any significant amount of native base pairs. Such a transition state supports a model in which the rate-limiting step of the hybridization reaction is the alignment of the two strands prior to base pairing.


Holographic Imaging Reveals the Mechanism of Wall Entrapment in Swimming Bacteria

Silvio Bianchi, Filippo Saglimbeni, and Roberto Di Leonardo

Self-propelled particles, both biological and synthetic, are stably trapped by walls and develop high concentration peaks over bounding surfaces. In swimming bacteria, like E. coli, the physical mechanism behind wall entrapment is an intricate mixture of hydrodynamic and steric interactions with a strongly anisotropic character. The building of a clear physical picture of this phenomenon demands direct and full three-dimensional experimental observations of individual wall entrapment events. Here, we demonstrate that, by using a combination of three-axis holographic microscopy and optical tweezers, it is possible to obtain volumetric reconstructions of individual E. coli cells that are sequentially released at a controlled distance and angle from a flat solid wall. We find that hydrodynamic couplings can slow down the cell before collision, but reorientation only occurs while the cell is in constant contact with the wall. In the trapped state, all cells swim with the average body axis pointing into the surface. The amplitude of this pitch angle is anticorrelated to the amplitude of wobbling, thus indicating that entrapment is dominated by near-field couplings between the cell body and the wall. Our approach opens the way to three-dimensional quantitative studies of a broad range of fast dynamical processes in motile bacteria and eukaryotic cells.


Improving Sensitivity and Reproducibility of SERS Sensing in Microenvironments Using Individual, Optically Trapped Surface-Enhanced Raman Spectroscopy(SERS) Probes

Pietro Strobbia, Adam Mayer, Brian M Cullum

Surface-enhanced Raman spectroscopy (SERS) sensors offer many advantages for chemical analyses, including the ability to provide chemical specific information and multiplexed detection capability at specific locations. However, to have operative SERS sensors for probing microenvironments, probes with high signal enhancement and reproducibility are necessary. To this end, dynamic enhancement of SERS (i.e., in-situ amplification of signal-to-noise and signal-to-background ratios) from individual probes has been explored. In this paper, we characterize the use of optical tweezers to amplify SERS signals as well as suppress background signals via trapping of individual SERS active probes. This amplification is achieved through a steady presence of a single “hot” particle in the focus of the excitation laser. In addition to increases in signal and concomitant decreases in non-SERS backgrounds, optical trapping results in an eightfold increase in the stability of the signal as well. This enhancement strategy was demonstrated using both single and multilayered SERS sub-micron probes, producing combined signal enhancements of 24-fold (beyond the native 106 SERS enhancement) for a three-layered geometry. The ability to dynamically control the enhancement offers the possibility to develop SERS-based sensors and probes with tailored sensitivities. In addition, since this trapping enhancement can be used to observe individual probes with low laser fluences, it could offer particular interest in probing the composition of microenvironments not amenable to tip-enhanced Raman spectroscopy or other scanning probe methods (e.g., intracellular analyses, etc.).


Effect of Extinction on Separation of Nanoparticle Enantiomers With Chiral Optical Forces

Semen A. Andronaki, Weiren Zhu, Mikhail Yu. Leonov, Alexey G. Shalkovskiy, Alexander V. Baranov, Anatoly V. Fedorov, Ivan D. Rukhlenko

Separation of enantiomers of chiral inorganic nanoparticles can be performed using enantioselective optical forces that are strong enough to make the ordered drift of nanoparticles faster than their movement down a concentration gradient. Here, we solve the problem of nanoparticle diffusion in a bounded domain in the presence of an exponentially decaying driving force, which can represent a chiral force exerted on nanoparticle enantiomers by a circularly polarized light beam exhibiting either scattering, absorption, or both. We analyze the steady state spatial distributions of two basic purity measures of chiral mixtures, showing that extinction puts a fundamental limit on the degree of enantiopurification achievable with optical forces. Our solution can be used to model resolution of racemates of any kind of chiral inorganic nanoparticles that strongly interact with light.


Tuesday, May 9, 2017

Energy Transfer between Colloids via Critical Interactions

Ignacio A. Martínez, Clemence Devailly, Artyom Petrosyan and Sergio Ciliberto

We report the observation of a temperature-controlled synchronization of two Brownian-particles in a binary mixture close to the critical point of the demixing transition. The two beads are trapped by two optical tweezers whose distance is periodically modulated. We notice that the motion synchronization of the two beads appears when the critical temperature is approached. In contrast, when the fluid is far from its critical temperature, the displacements of the two beads are uncorrelated. Small changes in temperature can radically change the global dynamics of the system. We show that the synchronisation is induced by the critical Casimir forces. Finally, we present the measure of the energy transfers inside the system produced by the critical interaction.


Forces between colloidal particles in aqueous solutions containing monovalent and multivalent ions

Gregor Trefalt, Thomas Palberg, Michal Borkovec

The present article provides an overview of the recent progress in the direct force measurements between individual pairs of colloidal particles in aqueous salt solutions. Results obtained by two different techniques are being highlighted, namely with the atomic force microscope (AFM) and optical tweezers. One finds that the classical theory of Derjaguin, Landau, Verwey, and Overbeek (DLVO) represents an accurate description of the force profiles even in the presence of multivalent ions, typically down to distances of few nanometers. However, the corresponding Hamaker constants and diffuse layer potentials must be extracted from the force profiles. At low salt concentrations, double layer forces remain repulsive and can be long ranged. At short distances, additional short range non-DLVO interactions may become important. Such an interaction is particularly relevant in the presence of multivalent counterions.


Folding behavior of a T-shaped, ribosome-binding translation enhancer implicated in a wide-spread conformational switch

My-Tra Le Wojciech K Kasprzak Taejin Kim Feng Gao Megan YL Young Xuefeng Yuan Bruce A Shapiro Joonil Seog Anne E Simon

Turnip crinkle virus contains a T-shaped, ribosome-binding, translation enhancer (TSS) in its 3’UTR that serves as a hub for interactions throughout the region. The viral RNA-dependent RNA polymerase (RdRp) causes the TSS/surrounding region to undergo a conformational shift postulated to inhibit translation. Using optical tweezers (OT) and steered molecular dynamic simulations (SMD), we found that the unusual stability of pseudoknotted element H4a/Ψ3 required five upstream adenylates, and H4a/Ψ3 was necessary for cooperative association of two other hairpins (H5/H4b) in Mg2+. SMD recapitulated the TSS unfolding order in the absence of Mg2+, showed dependence of the resistance to pulling on the 3D orientation and gave structural insights into the measured contour lengths of the TSS structure elements. Adenylate mutations eliminated one-site RdRp binding to the 3’UTR, suggesting that RdRp binding to the adenylates disrupts H4a/Ψ3, leading to loss of H5/H4b interaction and promoting a conformational switch interrupting translation and promoting replication.


Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity

Tjalle P. Hoekstra, Martin Depken, Szu-Ning Lin, Jordi Cabanas-Danés, Peter Gross, Remus T. Dame, Erwin J.G. Peterman, Gijs J.L. Wuite

DNA polymerase catalyzes the accurate transfer of genetic information from one generation to the next, and thus it is vitally important for replication to be faithful. DNA polymerase fulfills the strict requirements for fidelity by a combination of mechanisms: 1) high selectivity for correct nucleotide incorporation, 2) a slowing down of the replication rate after misincorporation, and 3) proofreading by excision of misincorporated bases. To elucidate the kinetic interplay between replication and proofreading, we used high-resolution optical tweezers to probe how DNA-duplex stability affects replication by bacteriophage T7 DNA polymerase. Our data show highly irregular replication dynamics, with frequent pauses and direction reversals as the polymerase cycles through the states that govern the mechanochemistry behind high-fidelity T7 DNA replication. We constructed a kinetic model that incorporates both existing biochemical data and the, to our knowledge, novel states we observed. We fit the model directly to the acquired pause-time and run-time distributions. Our findings indicate that the main pathway for error correction is DNA polymerase dissociation-mediated DNA transfer, followed by biased binding into the exonuclease active site. The number of bases removed by this proofreading mechanism is much larger than the number of erroneous bases that would be expected to be incorporated, ensuring a high-fidelity replication of the bacteriophage T7 genome.


Monday, May 8, 2017

Optical concatenation of a large number of beads with a single-beam optical tweezer

Remy Avila, Joaquín Ascencio-Rodríguez, Daniel Tapia-Merino, Oscar G. Rodríguez-Herrera, and Arturo González-Suárez

Optical tweezers consist of the spatial confinement of microscopic dielectric particles by the action of forces produced by the change in momentum of the photons of a highly focused laser beam that are deviated by the particle. In experiments that use a single laser beam, it is common to capture not only one but a few particles in the optical trap. However, to our knowledge, the formation of a long chain of beads optically confined with a single laser beam has never been reported. In this work, up to 73 silica spheres immersed in water are seen concatenated along the propagation direction of a 976-nm wavelength Gaussian laser of 300 mW of power. This long chain of beads is obtained when the laser is focused through an oil-immersion DIN microscope objective with 100× magnification and a numerical aperture of 1.25. When performing the same experiment using an infinity-corrected UplanFLN 100× objective with a numerical aperture of 1.3, the maximum number of concatenated beads is only 14. Our results suggest that the mechanisms responsible for the observed phenomena involve successive refocusing of the laser beam by each trapped sphere, optically induced dipole coupling (commonly referred to as optical binding), and aberrations generated by the DIN microscope objective.


Tunable nanophotonic array traps with enhanced force and stability

Fan Ye, Mohammad Soltani, James T. Inman, and Michelle D. Wang

A nanophotonic trapping platform based on on-chip tunable optical interference allows parallel processing of biomolecules and holds promise to make single molecule manipulation and precision measurements more easily and broadly available. The nanophotonic standing wave array trap (nSWAT) device [Nat. Nanotechnol. 9, 448 (2014); Nano Lett. 16, 6661 (2016)] represents such a platform and can trap a large array of beads by the evanescent field of the standing wave of a nanophotonic waveguide and reposition them using an integrated microheater. In this paper, by taking a systematic design approach, we present a new generation of nSWAT devices with significant enhancement of the optical trapping force, stiffness, and stability, while the quality of the standing wave trap is resistant to fabrication imperfections. The device is implemented on a silicon nitride photonic platform and operates at 1064 nm wavelength which permits low optical absorption by the aqueous solution. Such performance improvements open a broader range of applications based on these on-chip optical traps.


Volume Transitions of Isolated Cell Nuclei Induced by Rapid Temperature Increase

Chii J. Chan, Wenhong Li, Gheorghe Cojoc, Jochen Guck

Understanding the physical mechanisms governing nuclear mechanics is important as it can impact gene expression and development. However, how cell nuclei respond to external cues such as heat is not well understood. Here, we studied the material properties of isolated nuclei in suspension using an optical stretcher. We demonstrate that isolated nuclei regulate their volume in a highly temperature-sensitive manner. At constant temperature, isolated nuclei behaved like passive, elastic and incompressible objects, whose volume depended on the pH and ionic conditions. When the temperature was increased suddenly by even a few degrees Kelvin, nuclei displayed a repeatable and reversible temperature-induced volume transition, whose sign depended on the valency of the solvent. Such phenomenon is not observed for nuclei subjected to slow heating. The transition temperature could be shifted by adiabatic changes of the ambient temperature, and the magnitude of temperature-induced volume transition could be modulated by modifying the chromatin compaction state and remodeling processes. Our findings reveal that the cell nucleus can be viewed as a highly charged polymer gel with intriguing thermoresponsive properties, which might play a role in nuclear volume regulation and thermosensing in living cells.


Targeted Nanoparticle Thermometry: A Method to Measure Local Temperature at the Nanoscale Point Where Water Vapor Nucleation Occurs

Arwa A. Alaulamie, Susil Baral, Samuel C. Johnson, Hugh H. Richardson

An optical nanothermometer technique based on laser trapping, moving and targeted attaching an erbium oxide nanoparticle cluster is developed to measure the local temperature. The authors apply this new nanoscale temperature measuring technique (limited by the size of the nanoparticles) to measure the temperature of vapor nucleation in water. Vapor nucleation is observed after superheating water above the boiling point for degassed and nondegassed water. The average nucleation temperature for water without gas is 560 K but this temperature is lowered by 100 K when gas is introduced into the water. The authors are able to measure the temperature inside the bubble during bubble formation and find that the temperature inside the bubble spikes to over 1000 K because the heat source (optically-heated nanorods) is no longer connected to liquid water and heat dissipation is greatly reduced.


Radially dependent angular acceleration of twisted light

Jason Webster, Carmelo Rosales-Guzmán, and Andrew Forbes

While photons travel in a straight line at constant velocity in free space, the intensity profile of structured light may be tailored for acceleration in any degree of freedom. Here we propose a simple approach to control the angular acceleration of light. Using Laguerre–Gaussian modes as our twisted beams carrying orbital angular momentum, we show that superpositions of opposite handedness result in a radially dependent angular acceleration as they pass through a focus (waist plane). Due to conservation of orbital angular momentum, we find that propagation dynamics are complex despite the free-space medium: the outer part of the beam (rings) rotates in an opposite direction to the inner part (petals), and while the outer part accelerates, the inner part decelerates. We outline the concepts theoretically and confirm them experimentally. Such exotic structured light beams are topical due to their many applications, for instance in optical trapping and tweezing, metrology, and fundamental studies in optics.


Friday, May 5, 2017

Multispectral Optical Tweezers for Biochemical Fingerprinting of CD9-Positive Exosome Subpopulations

Randy P. Carney, Sidhartha Hazari, Macalistair Colquhoun, Di Tran, Billanna Hwang, Michael S. Mulligan, James D. Bryers, Eugenia Girda, Gary S. Leiserowitz, Zachary J. Smith, and Kit S. Lam

Extracellular vesicles (EVs), including exosomes, are circulating nanoscale particles heavily implicated in cell signaling and can be isolated in vast numbers from human biofluids. Study of their molecular profiling and materials properties is currently underway for purposes of describing a variety of biological functions and diseases. However, the large, and as yet largely unquantified, variety of EV subpopulations differing in composition, size, and likely function necessitates characterization schemes capable of measuring single vesicles. Here we describe the first application of multispectral optical tweezers (MS-OTs) to single vesicles for molecular fingerprinting of EV subpopulations. This versatile imaging platform allows for sensitive measurement of Raman chemical composition (e.g., variation in protein, lipid, cholesterol, nucleic acids), coupled with discrimination by fluorescence markers. For exosomes isolated by ultracentrifugation, we use MS-OTs to interrogate the CD9-positive subpopulations via antibody fluorescence labeling and Raman spectra measurement. We report that the CD9-positive exosome subset exhibits reduced component concentration per vesicle and reduced chemical heterogeneity compared to the total purified EV population. We observed that specific vesicle subpopulations are present across exosomes isolated from cell culture supernatant of several clonal varieties of mesenchymal stromal cells and also from plasma and ascites isolated from human ovarian cancer patients.


Optically driven full-angle sample rotation for tomographic imaging in digital holographic microscopy

Yu-chih Lin, Hui-Chi Chen, Han-Yen Tu, Chin-Yu Liu, and Chau-Jern Cheng

This study presents a novel tomographic imaging technique for living biomedical samples using an optically driven full-angle rotation scheme based on digital holographic microscopy, in which the three-dimensional refractive index distribution inside the sample can be measured and analyzed. To accomplish the full-angle sample rotation, two optical traps are driven by highly focused spots on the top and bottom of the sample. The rim image of the sample outside the focal depth at the different rotation angles and propagation distances can be corrected and compensated, respectively, via numerical focusing; therefore, tomographic imaging of the sample can be conducted. The proposed approach shows that an entire symmetric spectrum can be acquired for tomographic reconstruction without the missing apple core problem as in traditional sample-rotation schemes. The three-dimensional refractive index of living yeast in a fluid medium is measured and verified.


The chaperone toolbox at the single-molecule level: From clamping to confining

Mario J. Avellaneda, Eline J. Koers, Mohsin M. Naqvi, Sander J. Tans

Protein folding is well known to be supervised by a dedicated class of proteins called chaperones. However, the core mode of action of these molecular machines has remained elusive due to several reasons including the promiscuous nature of the interactions between chaperones and their many clients, as well as the dynamics and heterogeneity of chaperone conformations and the folding process itself. While troublesome for traditional bulk techniques, these properties make an excellent case for the use of single-molecule approaches. In this review, we will discuss how force spectroscopy, fluorescence microscopy, FCS, and FRET methods are starting to zoom in on this intriguing and diverse molecular toolbox that is of direct importance for protein quality control in cells, as well as numerous degenerative conditions that depend on it.


Nucleosome mobility and the regulation of gene expression: Insights from single-molecule studies

Sergei Rudnizky, Omri Malik, Adaiah Bavly, Lilach Pnueli, Philippa Melamed, Ariel Kaplan

Nucleosomes at the promoters of genes regulate the accessibility of the transcription machinery to DNA, and function as a basic layer in the complex regulation of gene expression. Our understanding of the role of the nucleosome's spontaneous, thermally driven position changes in modulating expression is lacking. This is the result of the paucity of experimental data on these dynamics, at high-resolution, and for DNA sequences that belong to real, transcribed genes. We have developed an assay that uses partial, reversible unzipping of nucleosomes with optical tweezers to repeatedly probe a nucleosome's position over time. Using the nucleosomes at the promoters of two model genes, Cga and Lhb, we show that the mobility of nucleosomes is modulated by the sequence of DNA and by the use of alternative histone variants, and describe how the mobility can affect transcription, at the initiation and elongation phases.


Simple and high efficient graded-index multimode fiber tweezers: simulation and experiment

Yaxun Zhang, Tong Wang, Zhihai Liu, Yu Zhang, Xiaoyun Tang, Enming Zhao, Xinghua Yang, Haili Jiang, Jianzhong Zhang, Jun Yang, and Libo Yuan

We propose and demonstrate a novel single fiber optical tweezer based on a graded-index multimode fiber (GIMMF), which works with a free length GIMMF (>30 cm). We achieve a three-dimensional stable trap of yeast cells by using the GIMMF optical tweezers. Compared with the single-mode fiber optical tweezers, the GIMMF optical tweezers possess large optical trapping forces. Owing to the freedom of the GIMMF length, the fabrication of the GIMMF optical tweezers is simple, repeatable, and highly efficient. The GIMMF tweezers have the penitential to become a new member of the single fiber optical tweezers family and have a wide range of applications in the medical and biological cytology fields.


Wednesday, May 3, 2017

Optical pressure and numerical simulation of optical forces

Olav Gaute Hellesø

Optical forces on a particle can be calculated using numerical methods and by integrating the Maxwell stress tensor over the surface of the particle. It is shown that this gives considerable numerical noise for the radiation force on particles with refractive index close to that of the surrounding medium and that a large number of mesh elements are necessary. It is found preferable to calculate the force from the local optical pressure, as this gives considerably less noise and requires significantly fewer mesh elements. Results are also compared with an analytical model based on Mie theory.


Observation of Nanoscale Refractive Index Contrast via Photoinduced Force Microscopy

Antonio Ambrosio, Robert Charles Devlin, Federico Capasso, and William L. Wilson

Near-field optical microscopy (NSOM) is a scanning probe technique that allows optical imaging of sample surfaces with nanoscale resolution. Generally, all NSOM schemes rely on illuminating the sample surface and collecting the localized scattered light resulting from the interaction of the microscopes nanoscale probe with the sample surface in the illuminated region. Currently, a new set of nanospectroscopic techniques are being developed using Atomic Force Microscopes to detect optical interactions without detecting any light. One of these approaches is photoinduced force microscopy (PiFM), where local optical forces, originated by the illumination of the tip–sample region, are mechanically detected as forced oscillations of the cantilever of an atomic force microscope operating in a multifrequency mode. In this article we show high resolution nanoimaging via PiFM with a contrast only related to the local refractive index of a sample specifically designed to unambiguously decouple morphology from optical response at the nanoscale. Imaging lateral resolution better than 10 nm is obtained, and the optimization of the contrast mechanism is described. Our results represent a step forward in understanding the potential of the PiFM technique, showing the possibility of high resolution imaging of the local polarizability of the sample and subsequently using the mechanism to explore complex spectral behavior at the nanoscale.


Direct measurement of microtubule attachment strength to yeast centrosomes

Kimberly K. Fong, Krishna K. Sarangapani, Erik C. Yusko, Michael Riffle, Aida Llauró, Beth Graczyk, Trisha N. Davis, and Charles L. Asbury

Centrosomes, or spindle pole bodies (SPBs) in yeast, are vital mechanical hubs that maintain load-bearing attachments to microtubules during mitotic spindle assembly, spindle positioning, and chromosome segregation. However, the strength of microtubule-centrosome attachments is unknown, and the possibility that mechanical force might regulate centrosome function has scarcely been explored. To uncover how centrosomes sustain and regulate force, we purified SPBs from budding yeast and used laser trapping to manipulate single attached microtubules in vitro. Our experiments reveal that SPB-microtubule attachments are extraordinarily strong, rupturing at forces about 4-fold higher than kinetochore attachments under identical loading conditions. Furthermore, removal of the calmodulin-binding site from the SPB component, Spc110, weakens SPB-microtubule attachment in vitro and sensitizes cells to increased SPB stress in vivo. These observations show that calmodulin binding contributes to SPB mechanical integrity and suggest that its removal may cause pole delamination and mitotic failure when spindle forces are elevated. We propose that the very high strength of SPB-microtubule attachments may be important for spindle integrity in mitotic cells, so that tensile forces generated at kinetochores do not cause microtubule detachment and delamination at SPBs.


Optical trapping-assisted SERS platform for chemical and biosensing applications: Design perspectives

Yufeng Yuan, Yining Lin, Bobo Gu, Nishtha Panwar, Swee Chuan Tjin, Jun Song, Junle Qu, Ken-Tye Yong

Useful approaches to tackle Brownian motion of micro/nanoparticles and overcome the poor reproducibility of surface-enhanced Raman scattering (SERS) events are highly desirable, especially for performing SERS sensing in ultra-low concentration of analytes. When integrated with the SERS measurement system, optical trapping is a versatile approach for manipulating particles and thereby improving the SERS performance. A review of the recent research advancements in optical trapping-assisted SERS platform can provide critical inputs for optimizing SERS-based sensing methods for real-life applications. In this paper, we present an in-depth review on the systematic classification of optical trapping-assisted (e.g., far-field and near-field optical tweezers) SERS sensing platform and discuss its latest practical applications in biosensing, bioimaging, chemical monitoring, particle manipulation, single cell analysis, etc. Also, we summarize some important strategies to suppress the plasmonic heating effect which hinders the stability of optical tweezers. Furthermore, we also propose non-optical trapping approaches for manipulating nanoparticles/molecules that are promising for prospective SERS sensing. For example, plasmonic heating is not completely deleterious to particle manipulation. Particularly, plasmon-enhanced thermophoresis technique is a useful non-optical approach for trapping particles/molecules and incorporating with SERS detection. Finally, we conclude with future perspectives for designing the new generation of optical tweezers.


Manipulation and Motion of Organelles and Single Molecules in Living Cells

Kamilla Norregaard, Ralf Metzler, Christine M. Ritter, Kirstine Berg-Sørensen, and Lene B. Oddershede

The biomolecule is among the most important building blocks of biological systems, and a full understanding of its function forms the scaffold for describing the mechanisms of higher order structures as organelles and cells. Force is a fundamental regulatory mechanism of biomolecular interactions driving many cellular processes. The forces on a molecular scale are exactly in the range that can be manipulated and probed with single molecule force spectroscopy. The natural environment of a biomolecule is inside a living cell, hence, this is the most relevant environment for probing their function. In vivo studies are, however, challenged by the complexity of the cell. In this review, we start with presenting relevant theoretical tools for analyzing single molecule data obtained in intracellular environments followed by a description of state-of-the art visualization techniques. The most commonly used force spectroscopy techniques, namely optical tweezers, magnetic tweezers, and atomic force microscopy, are described in detail, and their strength and limitations related to in vivo experiments are discussed. Finally, recent exciting discoveries within the field of in vivo manipulation and dynamics of single molecule and organelles are reviewed.


Tuesday, May 2, 2017

A Simple Trapping and Manipulation Method of Biological Cell Using Robot-Assisted Optical Tweezers: Singular Perturbation Approach

Xiang Li; Chien Chern Cheah

Optical tweezers have been widely utilized in biological sciences and biomedical engineering because of the attractive feature of manipulating microobjects without physical contacts. The integration of optical tweezers and robotic technologies has also led to the emergence of many robot-assisted optical manipulation systems, and a variety of motion control schemes have been reported to automated the process and improve the efficiency. However, the performance of existing control methods for optical tweezers is commonly limited by several issues: 1) presence of spatially varying trapping stiffness, which is difficult to model and identify; 2) requirements of high-order state variables, which are not measurable; and 3) effectiveness of trapping is only valid locally around the center of laser beam. These issues lead to the constructions of controllers, which are computationally involved. This paper presents a simple tracking control method for optical manipulation, which enables the laser beam to automatically trap and then manipulate the cell to track a time-varying trajectory, without high-order derivatives or construction of observers. The development of the proposed controller is based on the singular perturbation approach, by treating the fast manipulator or stage dynamics as a perturbation of the slow cell dynamics, such that the lowest control complexity is achieved. The exponential stability of the overall system that consists of the fast and slow subsystems is proved by using Tikhonov's theorem. Experimental results are presented to illustrate the performance of the proposed controller.

Analytical calculations of optical trapping forces for drag calibration: Effects of mismatch between beam focus and particle center

Jin Hyun Lim, Dong Woo Kang, Bum Jun Park

We present analytical calculations of optical forces for drag calibrations via the geometrical optics approximation (GOA) for the case in which a single laser beam is strongly focused on a polystyrene microsphere dispersed in an aqueous phase. When the beam focus is mismatched with the particle center owing to the presence of vertical forces, such as gravity, buoyancy, and the radiation force caused by the laser beam, the trapped particle would be displaced in a radial direction when the lateral drag force is applied. Based on the analytical calculations of optical trapping forces and the force balance upon dragging, we found that the critical laser power at which the beam focus matches the particle center exhibits a power-law relationship with the particle radius a according to the empirical expression log(Pcrit (mW)) = 2.899 log(2a (µm)) - 2.316. We also found an empirical expression for the mechanical equilibrium position of the vertical displacement Δzeq0(Δy=0) as functions of the laser power P and the particle radius, Δz0eq=[Fgb(pN)−0.068P(mW)][2.7a(μm)−0.252]P(mW)Δzeq0=[Fgb(pN)−0.068P(mW)][2.7a(μm)−0.252]P(mW) , where Fgb is the sum of the gravitational and buoyant forces.


Multicellular Biohybrid Materials: Probing the Interplay of Cells of Different Types Precisely Positioned and Constrained on 3D Wireframe-Like Microstructures

Maurizio R. Gullo, Shoji Takeuchi, Oliver Paul
Driven by the unbroken miniaturization trend in microtechnology, the development of smaller, yet reliable and efficient, highly integrated microsystems can benefit from inherent capabilities of biological cells. In particular, by featuring multiple types of cells, biohybrid systems exhibiting self-contained sensing and actuation capabilities can be conceived. To ensure the proper functioning of such multicellular biohybrid systems, the intended cell arrangement needs to be maintained over time. Microscaffolds designed for this purpose should therefore selectively guide or hinder cell migration. However, the basic cell-structure interactions governing the cell migration and extension processes are not yet fully understood. This paper explores these interactions and proposes a method for the fabrication of advanced multicellular biohybrid materials. The method is based on wireframe-like 3D microstructures onto which several types of cells are successfully positioned and arranged by optical manipulation. Experiments exploring cell dynamics reveal geometry-dependent maximal migration and extension distances. Microscaffolds designed on the basis of these characteristics can guide cell migration, trigger structure-contained cell growth, and maintain a predetermined cell arrangement. The methods reported herein therefore provide insight into cell assembly and migration on 3D microscaffolds, which is an essential early step towards advanced multicellular biohybrid materials.


Experimental demonstration of square Fresnel zone plate with chiral side lobes

A. Vijayakumar, B. Vinoth, Igor V. Minin, Joseph Rosen, Oleg V. Minin, and Chau-Jern Cheng

In this study, we introduce what we believe is a novel holographic optical element called a chiral square Fresnel zone plate (CSFZP). The chirality is imposed on a square Fresnel zone plate (SFZP) using a nonclassical technique by rotating the half-period zones relative to one another. The rotation of the half-period zones, in turn, twists the side lobes of the diffraction pattern without altering the focusing properties inherent to a SFZP. As a consequence, the beam profile is hybrid, consisting of a strong central Gaussian focal spot with gradient force similar to that generated by a lens and twisted side lobes with orbital angular momentum. The optical fields at the focal plane were calculated and found to possess a whirlpool-phase profile and a twisted intensity profile. Analysis of the field variation along the direction of propagation revealed a spiraling phase and amplitude distribution. Poynting vector plot of the fields revealed the presence of angular momentum in the regions of chiral side lobes. The phase of the CSFZPs were displayed on a phase-only reflective spatial light modulator and illuminated using a laser. The intensity patterns recorded in the experiment match the calculated ones, with a strong central focal spot and twisted side lobes. The beam pattern was implemented in an optical trapping experiment and was found to possess particle trapping capabilities.


Single-Crystalline Gold Nanowires Synthesized from Light-Driven Oriented Attachment and Plasmon-Mediated Self-Assembly of Gold Nanorods or Nanoparticles

Shang-Yang Yu, Hariyanto Gunawan, Shiao-Wen Tsai, Yun-Ju Chen, Tzu-Chen Yen & Jiunn-Woei Liaw

Through the light-driven geometrically oriented attachment (OA) and self-assembly of Au nanorods (NRs) or nanoparticles (NPs), single-crystalline Au nanowires (NWs) were synthesized by the irradiation of a linearly-polarized (LP) laser. The process was conducted in a droplet of Au colloid on a glass irradiated by LP near-infrared (e.g. 1064 nm and 785 nm) laser beam of low power at room temperature and atmospheric pressure, without any additive. The FE-SEM images show that the cross sections of NWs are various: tetragonal, pentagonal or hexagonal. The EDS spectrum verifies the composition is Au, and the pattern of X-ray diffraction identifies the crystallinity of NWs with the facets of {111}, {200}, {220} and {311}. We proposed a hypothesis for the mechanism that the primary building units are aligned and coalesced by the plasmon-mediated optical torque and force to form the secondary building units. Subsequently, the secondary building units undergo the next self-assembly, and so forth the tertiary ones. The LP light guides the translational and rotational motions of these building units to perform geometrically OA in the side-by-side, end-to-end and T-shaped manners. Consequently, micron-sized ordered mesocrystals are produced. Additionally, the concomitant plasmonic heating causes the annealing for recrystallizing the mesocrystals in water.