Wednesday, March 22, 2017

Combining FRET and optical tweezers to study RhoGTPases spatio-temporal dynamics upon local stimulation

Federico Iseppon, Luisa MR Napolitano, Vincent Torre, Dan Cojoc

Local stimulation with optical tweezers has been used to mimic natural stimuli that occur in biological processes such as cell migration or differentiation. Carriers (beads and lipid vesicles) with sizes down to 30 nm can be manipulated with a high spatial and temporal resolution: they are positioned with a sub-micrometric precision on a specific cell compartment and the beginning of the stimulation can be triggered with millisecond precision. RhoGTPases are a Ras-related family of proteins that regulate many different functions including cell polarity, microtubule dynamics and membrane transport pathways. Here we combine local stimulation with FRET microscopy to study RhoGTPases spatial and temporal activation following guidance cue local stimulation. We used two different vectors for local delivery: silica micro-beads and micro-sized lipid vesicles. The experimental methods associated with neuronal growth cone local stimulation are discussed in detail, as well as the analysis methods. Here we present a protocol that enables to study neuronal growth cone cytoskeleton rearrangements in response to a gradient of molecules in a way that better mimics physiological conditions, and it can be similarly applied to each secreted molecule involved in cell signaling.


Laser-accelerated self-assembly of colloidal particles at the water–air interface

Mincheng Zhong, Ziqiang Wang, and Yinmei Li

We experimentally demonstrate that optical tweezers can be used to accelerate the self-assembly of colloidal particles at a water–air interface in this Letter. The thermal flow induced by optical tweezers dominates the growth acceleration at the interface. Furthermore, optical tweezers are used to create a local growth peak at the growing front, which is used to study the preferential incorporation positions of incoming particles. The results show that the particles surfed with a strong Marangoni flow tend to fill the gap and smoothen the steep peaks. When the peak is smooth, the incoming particles incorporate the crystal homogeneously at the growing front.


On the motion control of microparticles by means of an electromagnetic field increasing with time for spectroscopic applications

A. Ch. Izmailov

The possibility of controlling the motion of microparticles by means of external electromagnetic fields (nonresonance laser radiation, in particular) that induce potential wells for such particles, which are characterized by fixed spatial distribution but deepen over time to a certain level, are analyzed. It is assumed that the particles are located in high vacuum and are affected by nondissipative external forces. Slowing down of relatively fast particles when they pass through the discussed potential wells is shown. Such slowing down of particles is demonstrated using a nonresonance laser beam with intensity increasing over time as an example. Specific features of particle dynamics in the electromagnetic fields under consideration in the case of a one-dimensional rectangular potential well are studied in detail based on simple analytical relations derived from the fundamental equations of classical mechanics. The methods of particle cooling and localization demonstrated in the present work can substantially increase spectroscopy resolution of various microparticles, including, under certain conditions, atoms and molecules.


Single-molecule mechanochemical characterization of E. coli pol III core catalytic activity

M. Nabuan Naufer, David A. Murison, Ioulia Rouzina, Penny J. Beuning, Mark C. Williams

Pol III core is the three-subunit subassembly of the E. coli replicative DNA polymerase III holoenzyme. It contains the catalytic polymerase subunit α, the 3′ → 5′ proofreading exonuclease ε, and a subunit of unknown function, θ. We employ optical tweezers to characterize pol III core activity on a single DNA substrate. We observe polymerization at applied template forces F < 25 pN and exonucleolysis at F > 30 pN. Both polymerization and exonucleolysis occur as a series of short bursts separated by pauses. For polymerization, the initiation rate after pausing is independent of force. In contrast, the exonucleolysis initiation rate depends strongly on force. The measured force and concentration dependence of exonucleolysis initiation fits well to a two-step reaction scheme in which pol III core binds bimolecularly to the primer-template junction, then converts at rate k2 into an exo-competent conformation. Fits to the force dependence of kinit show that exo initiation requires fluctuational opening of two base pairs, in agreement with temperature- and mismatch-dependent bulk biochemical assays. Taken together, our results support a model in which the pol and exo activities of pol III core are effectively independent, and in which recognition of the 3′ end of the primer by either α or ε is governed by the primer stability. Thus, binding to an unstable primer is the primary mechanism for mismatch recognition during proofreading, rather than an alternative model of duplex defect recognition.


Optical methods for measuring DNA folding

Adam D. Smith, Obinna A. Ukogu, Luka M. Devenica, Elizabeth D. White, Ashley R. Carter

One of the most important biological processes is the dynamic folding and unfolding of deoxyribonucleic acid (DNA). The folding process is crucial for DNA to fit within the boundaries of the cell, while the unfolding process is essential for DNA replication and transcription. To accommodate both processes, the cell employs a highly active folding mechanism that has been the subject of intense study over the last few decades. Still, many open questions remain. What are the pathways for folding or unfolding? How does the folding equilibrium shift? And, what is the energy landscape for a particular process? Here, we review these emerging questions and the in vitro, optical methods that have provided answers, introducing the topic for those physicists seeking to step into biology. Specifically, we discuss two iconic experiments for DNA folding, the tethered particle motion (TPM) experiment and the optical tweezers experiment.

Tuesday, March 21, 2017

Manipulating particles with light: radiation and gradient forces

David S Bradshaw and David L Andrews
The manipulation of matter with electromagnetic radiation is a capacity that has been known for over a century. However, the prominence of such optical effects only grew rapidly following the invention of optical tweezers in the 1980s. While both the original theory and the early trapping techniques are based on the radiation force, optical tweezing uses the gradient force. This paper aims to differentiate between these two clearly distinct types of optical forces, which are sometimes confused in the literature. We also discuss three completely separate forms of optical torque that can be applied to a particle, also due to an electromagnetic field. These involve the transfer of either spin or orbital angular momentum from the beam to the particle, depending on the character of the light, or the often overlooked alignment effect that can act on a cylindrical particle due to a gradient force.


Visual guide to optical tweezers

Isaac C D Lenton, Alexander B Stilgoe, Halina Rubinsztein-Dunlop and Timo A Nieminen

It is common to introduce optical tweezers using either geometric optics for large particles or the Rayleigh approximation for very small particles. These approaches are successful at conveying the key ideas behind optical tweezers in their respective regimes. However, they are insufficient for modelling particles of intermediate size and large particles with small features. For this, a full field approach provides greater insight into the mechanisms involved in trapping. The advances in computational capability over the last decade have led to better modelling and understanding of optical tweezers. Problems that were previously difficult to model computationally can now be solved using a variety of methods on modern systems. These advances in computational power allow for full field solutions to be visualised, leading to increased understanding of the fields and behaviour in various scenarios. In this paper we describe the operation of optical tweezers using full field simulations calculated using the finite difference time domain method. We use these simulations to visually illustrate various situations relevant to optical tweezers, from the basic operation of optical tweezers, to engineered particles and evanescent fields.


Controllable generation and manipulation of micro-bubbles in water with absorptive colloid particles by CW laser radiation

O. V. Angelsky, A. Ya. Bekshaev, P. P. Maksimyak, A. P. Maksimyak, S. G. Hanson, and S. M. Kontush

Micrometer-sized vapor-gas bubbles are formed due to local heating of a water suspension containing absorptive pigment particles of 100 nm diameter. The heating is performed by CW near-infrared (980 nm) laser radiation with controllable power, focused into a 100 μm spot within a 2 mm suspension layer. By changing the laser power, four regimes are realized: (1) bubble generation; (2) stable growth of the existing bubbles; (3) stationary existence of the bubbles and (4) the bubbles’ shrinkage and collapse. This behavior is interpreted based on the temperature conditions. The generation and evolution of single bubbles and ensembles of bubbles with controllable sizes and numbers is demonstrated. The bubbles are grouped within the laser-illuminated region and form quasi-ordered structures. They can easily be moved and transported controlled by the focal spot. The results are useful for applications associated with the precise manipulation, sorting and specific delivery in nano- and micro-engineering problems.


Strong plasmonic confinement and optical force in phosphorene pairs

Hua Lu, Yongkang Gong, Dong Mao, Xuetao Gan, and Jianlin Zhao

The plasmonic responses in the spatially separated phosphorene (single-layer black phosphorus) pairs are investigated, mainly containing the field enhancement, light confinement, and optical force. It is found that the strong anisotropic dispersion of black phosphorus gives rise to the direction-dependent symmetric and anti-symmetric plasmonic modes. Our results demonstrate that the symmetrical modes possess stronger field enhancement, higher light confinement, and larger optical force than the anti-symmetric modes in the nanoscale structures. Especially, the light confinement ratio and optical force for the symmetric mode along the armchair direction of black phosphorus can reach as high as >90% and >3000 pN/mW, respectively. These results may open a new door for the light manipulation at nanoscale and the design of black phosphorus based photonic devices.


Methods for Elucidation of DNA-Anticancer Drug Interactions and their Applications in the Development of New Drugs

Majus Misiak, Francesco Mantegazza, Giovanni L. Beretta

DNA damaging agents including anthracyclines, camptothecins and platinum drugs are among most frequently used drugs in the chemotherapeutic routine. Due to their relatively low selectivity for cancer cells, administration of these drugs is associated with adverse side effects, inherent genotoxicity with risk of developing secondary cancers. Development of new drugs, which could be spared of these drawbacks and characterize by improved antitumor efficacy, remains challenging yet vitally important task. These properties are in large part dictated by the selectivity of interaction between the drug and DNA and in this way the studies aimed at elucidating the complex interactions between ligand and DNA represent a key step in the drug development. Studies of the drug-DNA interactions encompass determination of DNA sequence specificity and mode of DNA binding as well as kinetic, dynamic and structural parameters of binding. Here, we consider the types of interactions between small molecule ligands and polynucleotides, how they are affected by DNA sequence and structure, and what is their significance for the antitumor activity. Based on this knowledge, we discuss the wide array of experimental techniques available to researchers for studying drug-DNA interactions, which include absorption and emission spectroscopies, NMR, magnetic and optical tweezers or atomic force microscopy. We show, using the clinical and experimental anticancer drugs as examples, how these methods provide various types of information and at the same time complement each other to provide full picture of drug- DNA interaction and aid in the development of new drugs.