Wednesday, March 14, 2018

Investigation of albumin-derived perfluorocarbon-based capsules by holographic optical trapping

Jannis Köhler, Jegor Ruschke, Katja Bettina Ferenz, Cemal Esen, Michael Kirsch, and Andreas Ostendorf

Albumin-derived perfluorocarbon-based capsules are promising as artificial oxygen carriers with high solubility. However, these capsules have to be studied further to allow initial human clinical tests. The aim of this paper is to provide and characterize a holographic optical tweezer to enable contactless trapping and moving of individual capsules in an environment that mimics physiological (in vivo) conditions most effectively in order to learn more about the artificial oxygen carrier behavior in blood plasma without recourse to animal experiments. Therefore, the motion behavior of capsules in a ring shaped or vortex beam is analyzed and optimized on account of determination of the optical forces in radial and axial direction. In addition, due to the customization and generation of dynamic phase holograms, the optical tweezer is used for first investigations on the aggregation behavior of the capsules and a statistical evaluation of the bonding in dependency of different capsule sizes is performed. The results show that the optical tweezer is sufficient for studying individual perfluorocarbon-based capsules and provide information about the interaction of these capsules for future use as artificial oxygen carriers.


Single-molecule manipulation and detection

Deyu Zhao, Siyun Liu, Ying Gao

Compared to conventional ensemble methods, studying macromolecules at single-molecule level can reveal extraordinary clear and even surprising views for a biological reaction. In the past 20 years, single-molecule techniques have been undergoing a very rapid development, and these cutting edge technologies have revolutionized the biological research by facilitating single-molecule manipulation and detection. Here we give a brief review about these advanced techniques, including optical tweezers, magnetic tweezers, atomic force microscopy (AFM), hydrodynamic flow-stretching assay, and single-molecule fluorescence resonance energy transfer (smFRET). We are trying to describe their basic principles and provide a few examples of applications for each technique. This review aims to give a rather introductory survey of single-molecule techniques for audiences with biological or biophysical background.


Single nanoparticle trapping based on on-chip nanoslotted nanobeam cavities

Daquan Yang, Fei Gao, Qi-Tao Cao, Chuan Wang, Yuefeng Ji, and Yun-Feng Xiao

Optical trapping techniques are of great interest since they have the advantage of enabling the direct handling of nanoparticles. Among various optical trapping systems, photonic crystal nanobeam cavities have attracted great attention for integrated on-chip trapping and manipulation. However, optical trapping with high efficiency and low input power is still a big challenge in nanobeam cavities because most of the light energy is confined within the solid dielectric region. To this end, by incorporating a nanoslotted structure into an ultracompact one-dimensional photonic crystal nanobeam cavity structure, we design a promising on-chip device with ultralarge trapping potential depth to enhance the optical trapping characteristic of the cavity. In this work, we first provide a systematic analysis of the optical trapping force for an airborne polystyrene (PS) nanoparticle trapped in a cavity model. Then, to validate the theoretical analysis, the numerical simulation proof is demonstrated in detail by using the three-dimensional finite element method. For trapping a PS nanoparticle of 10 nm radius within the air-slot, a maximum trapping force as high as 8.28 nN/mW and a depth of trapping potential as large as 1.15×105 𝑘B𝑇 mW−1 are obtained, where 𝑘B is the Boltzmann constant and 𝑇 is the system temperature. We estimate a lateral trapping stiffness of 167.17 pN·nm−1· mW−1 for a 10 nm radius PS nanoparticle along the cavity 𝑥-axis, more than two orders of magnitude higher than previously demonstrated on-chip, near field traps. Moreover, the threshold power for stable trapping as low as 0.087 μW is achieved. In addition, trapping of a single 25 nm radius PS nanoparticle causes a 0.6 nm redshift in peak wavelength. Thus, the proposed cavity device can be used to detect single nanoparticle trapping by monitoring the resonant peak wavelength shift. We believe that the architecture with features of an ultracompact footprint, high integrability with optical waveguides/circuits, and efficient trapping demonstrated here will provide a promising candidate for developing a lab-on-a-chip device with versatile functionalities.


Optical Aggregation of Gold Nanoparticles for SERS Detection of Proteins and Toxins in Liquid Environment: Towards Ultrasensitive and Selective Detection

Antonino Foti, Cristiano D'andrea, Valentina Villari, Norberto Micali, Maria Grazia Donato, Barbara Fazio, Onofrio Maria Maragò, Raymond Gillibert, Marc Lamy de La Chapelle, Pietro Giuseppe Gucciardi

Optical forces are used to aggregate plasmonic nanoparticles and create SERS-active hot spots in liquid. When biomolecules are added to the nanoparticles, high sensitivity SERS detection is accomplished. Here we tailor this methodology to detect catalase and hemoglobin, two Raman resonant biomolecules, at concentrations down to 10 nM and 1 pM. Subsequently, we study the SERS signal in Bovine Serum Albumin as a function of the concentration, finding a monotonic dependence that suggests the possibility of quantitative detection. Finally, by exploiting nanoparticles functionalized with specific aptamers, we obtain first results on the SERS detection of Ochratoxin A, a fungal toxin found in food commodities and wine. This represents a first step towards the addition of molecular specificity to this novel biosensor strategy.


Optical tweezers system for live stem cell organization at the single-cell level

Peifeng Jing, Yannan Liu, Ethan G. Keeler, Nelly M. Cruz, Benjamin S. Freedman, and Lih Y. Lin

Cell manipulation is one of the most impactful applications for optical tweezers, and derived from this promise, we demonstrate a new optical tweezers system for the study of cell adhesion and organization. This method utilizes photonic-crystal-enhanced optical tweezers to manipulate cells with low laser intensities. By doing so, it enables effective cell patterning and culturing within the conditions necessary for successful differentiation and colony formation of human pluripotent stem cells. To this end, the biocompatibility of plasma-treated parylene-C for cell culturing was studied, and a thorough characterization of cellular interactive forces was performed using this system. Furthermore, this study also demonstrates construction of patterned cell arrays at arbitrary positions with micrometer-scale precision.


Functional role of the type 1 pilus rod structure in mediating host-pathogen interactions

Caitlin N Spaulding, Henry Louis Schreiber IV, Weili Zheng, Karen W Dodson, Jennie E Hazen, Matt S Conover, Fengbin Wang, Pontus Svenmarker, Areli Luna-Rico, Olivera Francetic, Magnus Andersson, Scott Hultgren, Edward H Egelman

Uropathogenic E. coli (UPEC), which cause urinary tract infections (UTI), utilize type 1 pili, a chaperone usher pathway (CUP) pilus, to cause UTI and colonize the gut. The pilus rod, comprised of repeating FimA subunits, provides a structural scaffold for displaying the tip adhesin, FimH. We solved the 4.2 Å resolution structure of the type 1 pilus rod using cryo-electron microscopy. Residues forming the interactive surfaces that determine the mechanical properties of the rod were maintained by selection based on a global alignment of fimA sequences. We identified mutations that did not alter pilus production in vitro but reduced the force required to unwind the rod. UPEC expressing these mutant pili were significantly attenuated in bladder infection and intestinal colonization in mice. This study elucidates an unappreciated functional role for the molecular spring-like property of type 1 pilus rods in host-pathogen interactions and carries important implications for other pilus-mediated diseases.


Tuesday, March 6, 2018

Mechanics and statistics of the worm-like chain

Andrew Marantan, L. Mahadevan

The worm-like chain model is a simple continuum model for the statistical mechanics of a flexible polymer subject to an external force. We offer a tutorial introduction to it using three approaches. First, we use a mesoscopic view, treating a long polymer (in two dimensions) as though it were made of many groups of correlated links or “clinks,” allowing us to calculate its average extension as a function of the external force via scaling arguments. We then provide a standard statistical mechanics approach, obtaining the average extension by two different means: the equipartition theorem and the partition function. Finally, we work in a probabilistic framework, taking advantage of the Gaussian properties of the chain in the large-force limit to improve upon the previous calculations of the average extension.


A Protocol for Real-time 3D Single Particle Tracking

Shangguo Hou, Kevin Welsher
Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 µm2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.


An optical levitation system for a physics teaching laboratory

Oscar Isaksson, Magnus Karlsteen, Mats Rostedt and Dag Hanstorp

We describe an experimental system based on optical levitation of an oil droplet. When combined with an applied electric field and a source of ionizing radiation, the setup permits the investigation of physical phenomena such as radiation pressure, light diffraction, the motion of a charged particle in an oscillating electric field, and the interaction of ionizing radiation with matter. The trapping occurs by creating an equilibrium between a radiation pressure force and the force of gravity. We have found that an oil droplet can be trapped for at least nine hours. The system can be used to measure the size and total electric charge on the trapped droplet. The intensity of the light from the trapping laser that is scattered by the droplet is sufficient to allow the droplet to be easily seen with the naked eye, covered by laser alignment goggles. When oscillating under the influence of an ac electric field, the motion of the droplet can be described as that of a driven, damped harmonic oscillator. The magnitude and polarity of the charge can be altered by exposing the droplet to ionizing radiation from a low-activity radioactive source. Our goal was to design a hands-on setup that allows undergraduate and graduate students to observe and better understand fundamental physical processes.


Full molecular trajectories of RNA polymerase at single base-pair resolution

Maurizio Righini, Antony Lee, Cristhian Cañari-Chumpitaz, Troy Lionberger, Ronen Gabizon, Yves Coello, Ignacio Tinoco and Carlos Bustamante

Optical tweezers enable scientists to follow the dynamics of molecular motors at high resolution. The ability to discern a motor’s discrete steps reveals important insights on its operation. Some motors operate at the scale of angstroms, rendering the observation of their steps extremely challenging. In some cases, such small steps have been observed sporadically; however, the full molecular trajectories of steps and intervals between steps remain elusive due to instrumental noise. Here, we eliminate the main source of noise of most high-resolution dual-trap optical tweezers and developed both a single-molecule assay and a self-learning algorithm to uncover the full trajectories of such a motor: RNA polymerase. Using this method, a whole new set of experiments becomes possible.