Friday, May 25, 2018

Quantifying Local Molecular Tension Using Intercalated DNA Fluorescence

Graeme A. King, Andreas S. Biebricher, Iddo Heller, Erwin J. G. Peterman, and Gijs J. L. Wuite

The ability to measure mechanics and forces in biological nanostructures, such as DNA, proteins and cells, is of great importance as a means to analyze biomolecular systems. However, current force detection methods often require specialized instrumentation. Here, we present a novel and versatile method to quantify tension in molecular systems locally and in real time, using intercalated DNA fluorescence. This approach can report forces over a range of at least ∼0.5–65 pN with a resolution of 1–3 pN, using commercially available intercalating dyes and a general-purpose fluorescence microscope. We demonstrate that the method can be easily implemented to report double-stranded (ds)DNA tension in any single-molecule assay that is compatible with fluorescence microscopy. This is particularly useful for multiplexed techniques, where measuring applied force in parallel is technically challenging. Moreover, tension measurements based on local dye binding offer the unique opportunity to determine how an applied force is distributed locally within biomolecular structures. Exploiting this, we apply our method to quantify the position-dependent force profile along the length of flow-stretched DNA and reveal that stretched and entwined DNA molecules—mimicking catenated DNA structures in vivo—display transient DNA–DNA interactions. The method reported here has obvious and broad applications for the study of DNA and DNA–protein interactions. Additionally, we propose that it could be employed to measure forces in any system to which dsDNA can be tethered, for applications including protein unfolding, chromosome mechanics, cell motility, and DNA nanomachines.


Probing Position-Dependent Diffusion in Folding Reactions Using Single-Molecule Force Spectroscopy

Daniel A.N. Foster, Rafayel Petrosyan, Andrew G.T. Pyo, Armin Hoffmann, Feng Wang, Michael T. Woodside

Folding of proteins and nucleic acids involves a diffusive search over a multidimensional conformational energy landscape for the minimal-energy structure. When examining the projection of conformational motions onto a one-dimensional reaction coordinate, as done in most experiments, the diffusion coefficient D is generally position dependent. However, it has proven challenging to measure such position-dependence experimentally. We investigated the position-dependence of D in the folding of DNA hairpins as a simple model system in two ways: first, by analyzing the round-trip time to return to a given extension in constant-force extension trajectories measured by force spectroscopy, and second, by analyzing the fall time required to reach a given extension in force jump measurements. These methods yielded conflicting results: the fall time implied a fairly constant D, but the round-trip time implied variations of over an order of magnitude. Comparison of experiments with computational simulations revealed that both methods were strongly affected by experimental artifacts inherent to force spectroscopy measurements, which obscured the intrinsic position-dependence of D. Lastly, we applied Kramers’s theory to the kinetics of hairpins with energy barriers located at different positions along the hairpin stem, as a crude probe of D at different stem positions, and we found that D did not vary much as the barrier was moved along the reaction coordinate. This work underlines the difficulties faced when trying to deduce position-dependent diffusion coefficients from experimental folding trajectories.


Active Mechanics Reveal Molecular-Scale Force Kinetics in Living Oocytes

Wylie W. Ahmed, Étienne Fodor, Maria Almonacid, Matthias Bussonnier, Marie-Hélène Verlhac, Nir Gov, Paolo Visco, Frédéric van Wijland, Timo Betz

Active diffusion of intracellular components is emerging as an important process in cell biology. This process is mediated by complex assemblies of molecular motors and cytoskeletal filaments that drive force generation in the cytoplasm and facilitate enhanced motion. The kinetics of molecular motors have been precisely characterized in vitro by single molecule approaches, but their in vivo behavior remains elusive. Here, we study the active diffusion of vesicles in mouse oocytes, where this process plays a key role in nuclear positioning during development, and combine an experimental and theoretical framework to extract molecular-scale force kinetics (force, power stroke, and velocity) of the in vivo active process. Assuming a single dominant process, we find that the nonequilibrium activity induces rapid kicks of duration τ ∼ 300 μs resulting in an average force of F ∼ 0.4 pN on vesicles in in vivo oocytes, remarkably similar to the kinetics of in vitro myosin-V. Our results reveal that measuring in vivo active fluctuations allows extraction of the molecular-scale activity in agreement with single-molecule studies and demonstrates a mesoscopic framework to access force kinetics.


Pure nanodiamonds for levitated optomechanics in vacuum

A C Frangeskou, A T M A Rahman, L Gines, S Mandal, O A Williams, P F Barker and G W Morley
Optical trapping at high vacuum of a nanodiamond containing a nitrogen vacancy centre would provide a test bed for several new phenomena in fundamental physics. However, the nanodiamonds used so far have absorbed too much of the trapping light, heating them to destruction (above 800 K) except at pressures above ~10 mbar where air molecules dissipate the excess heat. Here we show that milling diamond of 1000 times greater purity creates nanodiamonds that do not heat up even when the optical intensity is raised above 700 GW m−2 below 5 mbar of pressure.


Metalens optical 3D-trapping and manipulating of nanoparticles

Yurii E Geints and Alexander A. Zemlyanov

A principal design of a single-beam optical trap is proposed based on the planar metalens assembled from an ordered array of dielectric microspheres arranged in a closely packed micro-assembly with a hollow center. By means of FDTD numerical simulation, we study in the detail the spatial structure of the optical field within the trap active zone and present an example of metalens-trap operation demonstrating the capturing and propulsion of a glass nanoparticle by the optical field.


Cell contraction induces long-ranged stress stiffening in the extracellular matrix

Yu Long Han, Pierre Ronceray, Guoqiang Xu, Andrea Malandrino, Roger D. Kamm, Martin Lenz, Chase P. Broedersz, and Ming Guo

Animal cells in tissues are supported by biopolymer matrices, which typically exhibit highly nonlinear mechanical properties. While the linear elasticity of the matrix can significantly impact cell mechanics and functionality, it remains largely unknown how cells, in turn, affect the nonlinear mechanics of their surrounding matrix. Here, we show that living contractile cells are able to generate a massive stiffness gradient in three distinct 3D extracellular matrix model systems: collagen, fibrin, and Matrigel. We decipher this remarkable behavior by introducing nonlinear stress inference microscopy (NSIM), a technique to infer stress fields in a 3D matrix from nonlinear microrheology measurements with optical tweezers. Using NSIM and simulations, we reveal large long-ranged cell-generated stresses capable of buckling filaments in the matrix. These stresses give rise to the large spatial extent of the observed cell-induced matrix stiffness gradient, which can provide a mechanism for mechanical communication between cells.


Thursday, May 24, 2018

High-precision joint amplitude and phase control of spatial light using a digital micromirror device

Lei Liu, Yesheng Gao, Xingzhao Liu

Control over both amplitude and phase of optical beams plays an important role in optics, especially in the field of optical trapping. Trapping particles needs optical beams with specified intensity distribution and desired phase distribution, so joint control over light beams with high precision is indispensable. In this paper, a novel joint amplitude and phase control method specially designed for high precision using a digital micromirror device (DMD) is proposed. An off-axis 4f-configuration and a pattern formation algorithm are required for the implementation of the proposed method. Getting DMD pattern by considering the correlations among all pixels guarantees the high precision but increases the computation complexity, which makes it difficult to realize. Thus we present a pattern formation algorithm based on mixed integer programming and its improved version with higher computing efficiency. Experimental tests have been conducted to verify the superior performance of the proposed method over the conventional methods, such as Lee holography and Superpixel method. Measured results and quantitative analyses show that the proposed method enables simultaneous and independent control over amplitude and phase of light beams with a higher precision and performs better when steepest phase gradients exist in the target field. What is more, vortex beam and line beam can be generated accurately by the proposed method.

Optical guiding-based cell focusing for Raman flow cell cytometer

Ravi Shanker Verma, Sunita Ahlawat and Abha Uppal

We report the use of an optical guiding arrangement generated in a microfluidic channel to produce a stream of single cells in a line for single-cell Raman spectroscopic analysis. The optical guiding arrangement consisted of dual-line optical tweezers, generated using a 1064 nm laser, aligned in the shape of a ‘Image ID:c8an00037a-u1.gif’ symbol. By controlling the laser power in the tweezers and the flow rate in the microfluidic channel, a single line flow of cells could be produced in the tail of the guiding arrangement, where the 514.5 nm Raman excitation beam was also located. Furthermore, by resonantly exciting the Raman spectrum, a good-quality Raman spectrum could be recorded from the flowing single cells as they passed through the Raman excitation focal spot without the need to trap the cells. As a proof of concept, it was shown that red blood cells (RBCs) could be guided to the tail of the optical guide and the Raman spectra of the resonantly excited cells could be recorded in a continuous manner without trapping the cells at a cell flow rate of ∼500 cells per h. From the recorded spectra, we were able to distinguish between RBCs containing hemoglobin in the normal form (normal-RBCs) and the met form (met-RBCs) from a mixture of RBCs comprising met-RBCs and normal-RBCs in a ratio of 1 : 9.


Optical trapping and manipulation of single particles in air: Principles, technical details, and applications

Zhiyong Gong, Yong-Le Pan, Gorden Videen, Chuji Wang

Trapping a single aerosol particle allows detailed investigation of its fundamental properties over extended time periods without external interferences. Optical trapping has developed into a powerful tool to perform such single-particle studies. However, trapping and manipulating a single particle in air, especially an irregularly shaped, absorbing particle, is much more challenging than that of a particle in a liquid solution. Even though the underlying mechanisms are not fully understood, recent experimental developments advanced the technique for trapping single particles in air, making it possible to manipulate and characterize a wide range of single particles. In this paper, we review recently demonstrated optical configurations for trapping and manipulating single airborne particles. Based on different trapping principles, we tentatively categorize them into radiation-pressure traps, photophoretic traps, and universal optical traps (UOTs). Radiation-pressure traps are based on the radiation pressure force resulting from photon momentum transfer; they include the early optical levitation configurations and the well-known optical tweezers. Photophoretic traps are based on the complex photophoretic forces that occur in absorbing particles; they are classified by the optical arrangements and include single-beam, dual-beam, and confocal-beam traps. UOTs can trap a variety of different types of particles, including transparent or absorbing, spherical or irregularly shaped, and liquid or solid particles. In order to evaluate each optical trapping scheme, four key aspects, i.e., simplicity, robustness, flexibility, and efficiency, of an optical trapping configuration are discussed. In addition to the stable optical trapping, optical manipulations from one dimension to three dimensions allow studying various single particles with great flexibility. With the single particle stably trapped and flexibly manipulated in air, other analytical techniques can be used to characterize these particles. Recent updates on optical methods for characterizing and monitoring single particles in air are discussed, such as light scattering, Raman spectroscopy, and cavity ringdown spectroscopy (CRDS).


Multiple optical trapping assisted bead-array based fluorescence assay of free and total prostate-specific antigen in serum

Di Cao, Cheng-Yu Li, Chu-Bo Qi, Hong-Lei Chen, Dai-Wen Pang, Hong-WuTang

Although suspension bead-based assay technology has been widely used owing to its advantages of high-throughput and microvolume detection, its sensitivity is greatly limited because it detects the fluorescence signal emitted by microbeads for a short time in the flowing fluid. In this work, we present the approach for prostate-specific antigen (PSA) detection of both free PSA (fPSA) and total PSA (tPSA) based on bead-array based fluorescence imaging by combining multiple optical trapping and bead-based bioassays. The polystyrene beads were employed to enrich the targets using the classic sandwich immuno-binding and tagged with fluorescent quantum dots (QDs), and the QDs-tagged beads in suspension were trapped array-by-array using multiple optical tweezers constructed with a diffraction optical element and excited with a 405 nm fiber laser for wide-field fluorescence imaging. The distinctive size information from the image of the trapped beads enabled the sorting of different targets. Moreover, the limits of detection for fPSA and tPSA are 3.8 pg/mL and 2.5 pg/mL respectively with good specificity. More importantly, this strategy was successfully used to detect fPSA and tPSA simultaneously in real serum samples. The high sensitivity, good selectivity, and tiny sample volume make this strategy a promising method for life sciences and clinical applications.