Friday, September 19, 2014

Direct Observation of Dynamic Mechanical Regulation of DNA Condensation by Environmental Stimuli

Amy Lee, Adam Karcz, Ryan Akman, Tai Zheng, Sara Kwon, Szu-Ting Chou, Sarah Sucayan, Dr. Lucas J. Tricoli, Jason M. Hustedt, Dr. Qixin Leng, Prof. Jason D. Kahn, Prof. A. James Mixson and Prof. Joonil Seog

Gene delivery is a promising way to treat hereditary diseases and cancer; however, there is little understanding of DNA:carrier complex mechanical properties, which may be critical for the protection and release of nucleic acids. We applied optical tweezers to directly measure single-molecule mechanical properties of DNA condensed using 19-mer poly-l-lysine (PLL) or branched histidine–lysine (HK) peptides. Force–extension profiles indicate that both carriers condense DNA actively, showing force plateaus during stretching and relaxation cycles. As the environment such as carrier concentration, pH, and the presence of zinc ions changes, DNA:HK complexes showed dynamically regulated mechanical properties at multiple force levels. The fundamental knowledge from this study can be applied to design a mechanically tailored complex which may enhance transfection efficiency by controlling the stability of the complex temporally and spatially.


Cell-cell proximity effects in multi-cell electroporation

Brian E. Henslee, Andrew Morss, Xin Hu, Gregory P. Lafyatis and L. James Lee

We report a fundamental study of how the electropermeabilization of a cell is affected by nearby cells. Previous researchers studying electroporation of dense suspensions of cells have observed, both theoretically and experimentally, that such samples cannot be treated simply as collections of independent cells. However, the complexity of those systems makes quantitative modeling difficult. We studied the change in the minimum applied electric field, the threshold field, required to affect electropermeabilization of a cell due to the presence of a second cell. Experimentally, we used optical tweezers to accurately position two cells in a custom fluidic electroporation device and measured the threshold field for electropermeabilization. We also captured video of the process. In parallel, finite element simulations of the electrostatic potential distributions in our systems were generated using the 3-layer model and the contact resistance methods. Reasonably good agreement with measurements was found assuming a model in which changes in a cell's threshold field were predicted from the calculated changes in the maximum voltage across the cell's membrane induced by the presence of a second cell. The threshold field required to electroporate a cell is changed ∼5%–10% by a nearby, nearly touching second cell. Cells aligned parallel to the porating field shield one another. Those oriented perpendicular to the field enhance the applied field's effect. In addition, we found that the dynamics of the electropermeabilization process are important in explaining observations for even our simple two-cell system.


Probing the coupled adhesion and deformation characteristics of suspension cells

T. H. Hui, Q. Zhu, Z. L. Zhou, J. Qian and Y. Lin

By combining optical trapping with fluorescence imaging, the adhesion and deformation characteristics of suspension cells were probed on single cell level. We found that, after 24 h of co-culturing, stable attachment between non-adherent K562 cells and polystyrene beads coated with fibronectin, collagen I, or G-actin can all be formed with an adhesion energy density in the range of 1–3×10−2 mJ/m2, which is about one order of magnitude lower than the reported values for several adherent cells. In addition, it was observed that the formation of a stronger adhesion is accompanied with the appearance of a denser actin cell cortex, especially in the region close to the cell-bead interface, resulting in a significant increase in the apparent modulus of the cell. Findings here could be important for our understanding of why the aggregation of circulating cells, like that in leukostasis, takes place in vivo as well as how such clusters of non-adherent cells behave. The method proposed can also be useful in investigating adhesion and related phenomena for other cell types in the future.


A Quantitative Comparison of Single-Cell Whole Genome Amplification Methods

Charles F. A. de Bourcy, Iwijn De Vlaminck, Jad N. Kanbar, Jianbin Wang, Charles Gawad, Stephen R. Quake

Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA) is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA), Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and the PicoPLEX single-cell WGA kit (NEB-WGA). We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.


Single-beam three-dimensional optical trapping at extremely low insertion angles via optical fiber optimization

Steven Ross; Mark F. Murphy; Francis Lilley; Michael J. Lalor; David R. Burton

Employing optical fiber to deliver the trapping laser to the sample chamber significantly reduces the size and costs of optical tweezers (OT). The utilization of fiber decouples the OT from the microscope, providing scope for system portability, and the potential for uncomplicated integration with other advanced microscopy systems. For use with an atomic force microscope, the fiber must be inserted at an angle of 10 deg to the plane of the sample chamber floor. However, the literature states that optical trapping with a single fiber inserted at an angle ≤20  deg is not possible. This paper investigates this limitation and proposes a hypothesis that explains it. Based on this explanation, a tapered-fiber optical tweezer system is developed. This system demonstrates that such traps can indeed be made to function in three-dimensions (3-D) at insertion angles of ≤10  deg using relatively low optical powers, provided the fiber taper is optimized. Three such optimized tapered fiber tips are presented, and their ability to optically trap both organic and inanimate material in 3-D is demonstrated. The near-horizontal insertion angle introduced a maximum trapping range (MTR). The MTR of the three tips is determined empirically, evaluated against simulated data, and found to be tunable through taper optimization.


Effects of Coating on the Optical Trapping Efficiency of Microspheres via Geometrical Optics Approximation

Bum Jun Park and Eric M. Furst

We present the optical trapping forces that are generated when a single laser beam strongly focuses on a coated dielectric microsphere. On the basis of geometrical optics approximation (GOA), in which a particle intercepts all of the rays that make up a single laser beam, we calculate the trapping forces with varying coating thickness and refractive index values. To increase the optical trapping efficiency, the refractive index (nb) of the coating is selected such that na < nb < nc, where na and nc are the refractive indices of the medium and the core material, respectively. The thickness of the coating also increases trapping efficiency. Importantly, we find that trapping forces for the coated particles are predominantly determined by two rays: the incident ray and the first refracted ray to the medium.


Mechanical Detection of a Long-Range Actin Network Emanating from a Biomimetic Cortex

Matthias Bussonnier, Kevin Carvalho, Joël Lemière, Jean-François Joanny, Cécile Sykes, Timo Betz

Actin is ubiquitous globular protein that polymerizes into filaments and forms networks that participate in the force generation of eukaryotic cells. Such forces are used for cell motility, cytokinesis, and tissue remodeling. Among those actin networks, we focus on the actin cortex, a dense branched network beneath the plasma membrane that is of particular importance for the mechanical properties of the cell. Here we reproduce the cellular cortex by activating actin filament growth on a solid surface. We unveil the existence of a sparse actin network that emanates from the surface and extends over a distance that is at least 10 times larger than the cortex itself. We call this sparse actin network the “actin cloud” and characterize its mechanical properties with optical tweezers. We show, both experimentally and theoretically, that the actin cloud is mechanically relevant and that it should be taken into account because it can sustain forces as high as several picoNewtons (pN). In particular, it is known that in plant cells, actin networks similar to the actin cloud have a role in positioning the nucleus; in large oocytes, they play a role in driving chromosome movement. Recent evidence shows that such networks even prevent granule condensation in large cells.


Numerical study of nanoparticle sensors based on the detection of the two-photon-induced luminescence of gold nanorod antennas

Zaoshan Huang, Qiaofeng Dai, Sheng Lan, Shaolong Tie

We investigate numerically the modification of the nonlinear optical properties of a nanoantenna in the trapping of nanoparticles (NPs) by using both the discrete dipole approximation method and the finite-difference time-domain technique. The nanoantenna, which is formed by two gold nanorods (GNRs) aligned end to end and separated by a small gap, can emit strong two-photon-induced luminescence (TPL) under the excitation of a femtosecond laser light which is resonant with its longitudinal surface plasmon resonance. In addition, the excited antenna can stably trap small NPs which in turn induce modifications in the emitted TPL. These two features make it a promising candidate for building highly sensitive detectors for NPs of different materials and sizes. It is demonstrated that sensors built with antennas possess higher sensitivities than those built with single GNRs and nanorod-based antennas are more sensitive than nanoprism-based antennas. In addition, it is found that the trapping probability for a second NP is significantly reduced for the antenna with a trapped NP, implying that trapping of NPs may occur sequentially. A relationship between the TPL of the system (antenna + NP) and the optical potential energy of the NP is established, enabling the extraction of the information on the optical potential energy and optical force by recording the TPL of the system. It is shown that the sequential trapping and releasing of NPs flowing in a microfluid channel can be realized by designing two different antennas arranged closely.