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.