## Wednesday, March 28, 2012

### Towards controlling molecular motions in fluorescence microscopy and optical trapping: a spatiotemporal approach

Arijit Kumar Deabc & Debabrata Goswami

This account reviews some recent studies pursued in our group on several control experiments with important applications in (one-photon) confocal and two-photon fluorescence laser-scanning microscopy and optical trapping with laser tweezers. We explore the simultaneous control of internal and external (i.e. centre-of-mass motion) degrees of freedom, which require the coupling of various control parameters to result in the spatiotemporal control. Of particular interest to us is the implementation of such control schemes in living systems. A live cell is a system of a large number of different molecules which combine and interact to generate complex structures and functions. These combinations and interactions of molecules need to be choreographed perfectly in time and space to achieve intended intra-cellular functions. Spatiotemporal control promises to be a versatile tool for dynamical control of spatially manipulated bio-molecules.

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### Optical trapping at low numerical aperture

S. Stallinga

A theory of optical trapping at low Numerical Aperture (NA) is presented. The theory offers an analytical description of the competition between the stabilizing gradient and destabilizing scattering force. The trade-off can be characterized by a single dimensionless trapping parameter, which increases with bead size to wavelength ratio $a/\lambda$ and refractive index contrast $m$ and decreases with NA. The gradient force dominates for small trapping parameters, the scattering force for large trapping parameters. The potential well depth, maximum forces and trap stiffness as a function of the three parameters ($a/\lambda$, $m$, NA) can be mapped onto universal functions of the trapping parameter. These functions do not depend on any free parameter. The universal well depth and maximum force curves match with numerical results based on the exact multipole expansion of the optical trapping force. The paraxial limit of low NA is relevant for compact optical tweezers based on Optical Pickup Units known from optical data storage.

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### Power spectrum and Allan variance methods for calibrating single-molecule video-tracking instruments

Bob M. Lansdorp and Omar A. Saleh

Single-molecule manipulation instruments, such as optical traps and magnetic tweezers, frequently use video tracking to measure the position of a force-generating probe. The instruments are calibrated by comparing the measured probe motion to a model of Brownian motion in a harmonic potential well; the results of calibration are estimates of the probe drag, α, and spring constant, κ. Here, we present both time- and frequency-domain methods to accurately and precisely extract α and κ from the probe trajectory. In the frequency domain, we discuss methods to estimate the power spectral density (PSD) from data (including windowing and blocking), and we derive an analytical formula for the PSD which accounts both for aliasing and the filtering intrinsic to video tracking. In the time domain, we focus on the Allan variance (AV): we present a theoretical equation for the AV relevant to typical single-molecule setups and discuss the optimal manner for computing the AV from experimental data using octave-sampled overlapping bins. We show that, when using maximum-likelihood methods to fit to the data, both the PSD and AV approaches can extract α and κ in an unbiased and low-error manner, though the AV approach is simpler and more robust.

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## Tuesday, March 20, 2012

### Direct observation of multiple misfolding pathways in a single prion protein molecule

Hao Yu, Xia Liu, Krishna Neupane, Amar Nath Gupta, Angela M. Brigley, Allison Solanki, Iveta Sosova, and Michael T. Woodside

Protein misfolding is a ubiquitous phenomenon associated with a wide range of diseases. Single-molecule approaches offer a powerful tool for deciphering the mechanisms of misfolding by measuring the conformational fluctuations of a protein with high sensitivity. We applied single-molecule force spectroscopy to observe directly the misfolding of the prion protein PrP, a protein notable for having an infectious misfolded state that is able to propagate by recruiting natively folded PrP. By measuring folding trajectories of single PrP molecules held under tension in a high-resolution optical trap, we found that the native folding pathway involves only two states, without evidence for partially folded intermediates that have been proposed to mediate misfolding. Instead, frequent but fleeting transitions were observed into off-pathway intermediates. Three different misfolding pathways were detected, all starting from the unfolded state. Remarkably, the misfolding rate was even higher than the rate for native folding. A mutant PrP with higher aggregation propensity showed increased occupancy of some of the misfolded states, suggesting these states may act as intermediates during aggregation. These measurements of individual misfolding trajectories demonstrate the power of single-molecule approaches for characterizing misfolding directly by mapping out nonnative folding pathways.

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### Hepatitis B surface antigen–antibody interactions studied by optical tweezers

Z.L. Zhou, B. Tang, A.H.W. Ngan, Z.N. Dong, and Y.S. Wu

The protein–protein interactions between hepatitis B surface antigen (HBsAg) and itsantibodies (anti-HBs) were studied by measuring the binding force between microspheres coated with such proteins using optical tweezers. The interaction force between the protein-coated microspheres was found to be strongly influenced by the acidity of the surrounding liquid medium, as well as the experimental temperature, and it reaches a maximum value at around pH 7.5 and temperature around 37°C. By measuring the protein distribution on the surfaces of the microspheres and their contact areas using scanning electron microscopy, the specific binding force between an HBsAg and anti-HBs protein pair is estimated to be around 4.8 pN at the optimum pH value and temperature at an applied loading rate of around 1 pN/s.

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### Three dimensional optical twisters-driven helically stacked multi-layered microrotors

Jolly Xavier, Raktim Dasgupta, Sunita Ahlawat, Joby Joseph, and Pradeep Kumar Gupta

We demonstrate tunable helically stacked multi-layered microrotors realized in vortex-embedded three dimensional (3D) optical twister patterns. Intensity-tunable annular irradiance profiles with higher order vortex are generated as well as simultaneously unfolded by phase-engineered multiple plane wave interference. In the individually tunable 3D helical bright arms of these unfolded vortex structures, 2 μm silica beads are optically trapped as spiraling multilayered handles of multi-armed microrotors. Further, multiple rows of such microrotors are parallelly actuated with controllable sense of rotation. We also present our observation on helical 3D stacking of micro-particles in these longitudinally gyrating multi-armed rotor traps.

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## Friday, March 16, 2012

### Reconfigurable interactions and three-dimensional patterning of colloidal particles and defects in lamellar soft media

Rahul P. Trivedi, Ivan I. Klevets, Bohdan Senyuk, Taewoo Lee, and Ivan I. Smalyukh

Colloidal systems find important applications ranging from fabrication of photonic crystals to direct probing of phenomena typically encountered in atomic crystals and glasses. New applications—such as nanoantennas, plasmonic sensors, and nanocircuits—pose a challenge of achieving sparse colloidal assemblies with tunable interparticle separations that can be controlled at will. We demonstrate reconfigurable multiscale interactions and assembly of colloids mediated by defects in cholesteric liquid crystals that are probed by means of laser manipulation and three-dimensional imaging. We find that colloids attract via distance-independent elastic interactions when pinned to the ends of cholesteric oily streaks, line defects at which one or more layers are interrupted. However, dislocations and oily streaks can also be optically manipulated to induce kinks, allowing one to lock them into the desired configurations that are stabilized by elastic energy barriers for structural transformation of the particle-connecting defects. Under the influence of elastic energy landscape due to these defects, sublamellar-sized colloids self-assemble into structures mimicking the cores of dislocations and oily streaks. Interactions between these defect-embedded colloids can be varied from attractive to repulsive by optically introducing dislocation kinks. The reconfigurable nature of defect–particle interactions allows for patterning of defects by manipulation of colloids and, in turn, patterning of particles by these defects, thus achieving desired colloidal configurations on scales ranging from the size of defect core to the sample size. This defect-colloidal sculpturing may be extended to other lamellar media, providing the means for optically guided self-assembly of mesoscopic composites with predesigned properties.

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### Initiation factor 2, tRNA, and 50S subunits cooperatively stabilize mRNAs on the ribosome during initiation

Tomoaki Masuda, Alexey N. Petrov, Ryo Iizuka, Takashi Funatsu, Joseph D. Puglisi, and Sotaro Uemura

Initiation factor 2 (IF2) is a key factor in initiation of bacterial protein synthesis. It recruits initiator tRNA to the small ribosomal subunit and facilitates joining of the large ribosomal subunit. Using reconstituted translation system of Escherichia coli and optical tweezers, we directly measure the rupture force between single ribosomal complexes and mRNAs for initiation complexes in the presence and the absence of IF2. We demonstrate that IF2 together with codon recognition by initiator tRNA increases the force required to dislocate mRNA from the ribosome complexes; mRNA stabilization by IF2 required the presence of a joined 50S subunit, and was independent of bound guanine nucleotide. IF2 thus helps lock the 70S ribosome over the start codon during initiation, thus maintaining reading frame. Our results show how mRNA is progressively stabilized on the ribosome through distinct steps of initiation.

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### Two independent switches regulate cytoplasmic dynein’s processivity and directionality

Wilhelm J. Walter, Michael P. Koonce, Bernhard Brenner, and Walter Steffen

Cytoplasmic dynein is a microtubule-based molecular motor that participates in a multitude of cell activities, from cell division to organelle transport. Unlike kinesin and myosin, where different tasks are performed by highly specialized members of these superfamilies, a single form of the dynein heavy chain is utilized for different functions. This versatility demands an extensive regulation of motor function. Using an improved application of an optical trap, we were now able to demonstrate that cytoplasmic dynein can generate a discrete power stroke as well as a processive walk in either direction; i.e., towards the plus- or towards the minus-end of a microtubule. Thus, dynein’s motor functions can be described by four basic modes of motion: processive and nonprocessive movement, and movement in the forward and reverse directions. Importantly, these four modes of movement can be controlled by two switches. One switch, based on phosphate, determines the directionality of movement. The second switch, depending on magnesium, converts cytoplasmic dynein from a nonprocessive to a processive motor. The two switches can be triggered separately or jointly by changing concentrations of phosphate and magnesium in the local environment. The control of four modes of movement by two switches has major implications for our understanding of the cellular functions and regulation of cytoplasmic dynein. Based on recent studies of dynein’s structure we are able to draw new conclusions on cytoplasmic dynein’s stepping mechanism.

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## Thursday, March 15, 2012

### Effects of viscosity on sperm motility studied with optical tweezers

Nicholas Hyun, Charlie Chandsawangbhuwana, Collin Yang-Wong, Michael W. Berns, Qingyuan Zhu, Linda Z. Shi

The purpose of this study is to analyze human sperm motility and energetics in media with different viscosities. Multiple experiments were performed to collect motility parameters using customized computer tracking software that measures the curvilinear velocity (VCL) and the minimum laser power (Pesc) necessary to hold an individual sperm in an optical trap. The Pesc was measured by using a 1064 nm Nd:YVO4continuous wave laser that optically traps motile sperm at a power of 450 mW in the focused trap spot. The VCL was measured frame by frame before trapping. In order to study sperm energetics under different viscous conditions sperm were labeled with the fluorescent dye DiOC6(3) to measure membrane potentials of mitochondria in the sperm midpiece. Fluorescence intensity was measured before and during trapping. The results demonstrate a decrease in VCL but an increase in Pesc with increasing viscosity. Fluorescent intensity is the same regardless of the viscosity level indicating no change in sperm energetics. The results suggest that, under the conditions tested, viscosity physically affects the mechanical properties of sperm motility rather than the chemical pathways associated with energetics.

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## Wednesday, March 14, 2012

### Sensing with Multipolar Second Harmonic Generation from Spherical Metallic Nanoparticles

Jérémy Butet, Isabelle Russier-Antoine, Christian Jonin, Noëlle Lascoux, Emmanuel Benichou, and Pierre-François Brevet

We show that sensing in the nonlinear optical regime using multipolar surface plasmon resonances is more sensitive in comparison to sensing in the linear optical regime. Mie theory, and its extension to the second harmonic generation from a metallic nanosphere, is used to describe multipolar second harmonic generation from silver metallic nanoparticles. The standard figure of merit of a potential plasmonic sensor based on this principle is then calculated. We finally demonstrate that such a sensor is more sensitive to optical refraction index changes occurring in the vicinity of the metallic nanoparticle than its linear counterpart.

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### Optical trapping with “on-demand” two-photon luminescence using Cr:LiSAF laser with optically addressed saturable Bragg reflector

Vasili G. Savitski, Nikolaus K. Metzger, Stephane Calvez, David Burns, W. Sibbett, and C. T. A. Brown

We demonstrate a diode-pumped Cr:LiSAF laser with controllable and reliable fast switching between its continuous-wave and mode-locked states of operation using an optically-addressed semiconductor Bragg reflector, permitting dyed microspheres to be continuously trapped and monitored using a standard microscope imaging and on-demand two-photon-excited luminescence techniques.

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## Tuesday, March 13, 2012

### Holographic optical bottle beams

Christina Alpmann, Michael Esseling, Patrick Rose, and Cornelia Denz

We present a convolution approach for the generation of optical bottle beams that combines established techniques of holographic optical trapping with hollow intensity distributions in order to manipulate absorbing particles. The versatility of our method is demonstrated by the simultaneous stable trapping of multiple particles at defined positions. Furthermore, the presented phase shaping technique allows for the dynamic manipulation of absorbing particles along arbitrary paths.

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## Monday, March 12, 2012

### Optical trapping via guided resonance modes in a Slot-Suzuki-phase photonic crystal lattice

Jing Ma, Luis Javier Martínez, and Michelle L. Povinelli
A novel photonic crystal lattice is proposed for trapping a two-dimensional array of particles. The lattice is created by introducing a rectangular slot in each unit cell of the Suzuki-Phase lattice to enhance the light confinement of guided resonance modes. Large quality factors on the order of 10^5 are predicted in the lattice. A significant decrease of the optical power required for optical trapping can be achieved compared to our previous design.

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### Assembly of microparticles by optical trapping with a photonic crystal nanocavity

C. Renaut, J. Dellinger, B. Cluzel, T. Honegger, D. Peyrade, E. Picard, F. de Fornel, and E. Hadji

In this work, we report the auto-assembly experiments of micrometer sized particles by optical trapping in the evanescent field of a photonic crystal nanocavity. The nanocavity is inserted inside an optofluidic cell designed to enable the real time control of the nanoresonator transmittance as well as the real time visualization of the particles motion in the vicinity of the nanocavity. It is demonstrated that the optical trap above the cavity enables the assembly of multiple particles in respect of different stable conformations.

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## Tuesday, March 6, 2012

### Optical Manipulation of Symbiotic Chlorella in Paramecium Bursaria Using a Fiber Axicon Microlens

K Taguchi, S Hirota, H Nakayama, D Kunugihara and Y Mihara

In this paper, chemically etched axicon fiber was proposed for laser trapping of symbiotic chlorella from paramecium bursaria. We fabricated axicon micro lenses on a single-mode bare optical fiber by selective chemical etching technique. The laser beam from fiber axicon microlens was strongly focused and optical forces were sufficient to move a symbiotic chlorella. From experimental results, it was found that our proposed fiber axicon microlens was a promising tool for cell trapping without physical contact.

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### Three-Dimensional Optical Trapping for Cell Isolation Using Tapered Fiber Probe by Dynamic Chemical Etching

K Taguchi, J Okada, Y Nomura and K Tamura

In this paper, chemically etched fiber probe was proposed for laser trapping and manipulation of cells. We fabricated tapered fiber probe by dynamic chemical etching technique. Three-Dimensional optical trap of a yeast cell dispersed in water solution could be formed by the fiber tip with 17deg tip. Optical forces were sufficient to move the yeast cell for trapping and manipulation. From these experimental results, it was found that our proposed tapered fiber tip was a promising tool for cell isolation.

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### Electrostatics of Nucleic Acid Folding under Conformational Constraint

Peter C. Anthony, Adelene Y. L. Sim, Vincent B. Chu, Sebastian Doniach, Steven M. Block, and Daniel Herschlag

RNA folding is enabled by interactions between the nucleic acid and its ion atmosphere, the mobile sheath of aqueous ions that surrounds and stabilizes it. Understanding the ion atmosphere requires the interplay of experiment and theory. However, even an apparently simple experiment to probe the ion atmosphere, measuring the dependence of DNA duplex stability upon ion concentration and identity, suffers from substantial complexity, because the unfolded ensemble contains many conformational states that are difficult to treat accurately with theory. To minimize this limitation, we measured the unfolding equilibrium of a DNA hairpin using a single-molecule optical trapping assay, in which the unfolded state is constrained to a limited set of elongated conformations. The unfolding free energy increased linearly with the logarithm of monovalent cation concentration for several cations, such that smaller cations tended to favor the folded state. Mg2+ stabilized the hairpin much more effectively at low concentrations than did any of the monovalent cations. Poisson–Boltzmann theory captured trends in hairpin stability measured for the monovalent cation titrations with reasonable accuracy, but failed to do so for the Mg2+ titrations. This finding is consistent with previous work, suggesting that Poisson–Boltzmann and other mean-field theories fail for higher valency cations where ion–ion correlation effects may become significant. The high-resolution data herein, because of the straightforward nature of both the folded and the unfolded states, should serve as benchmarks for the development of more accurate electrostatic theories that will be needed for a more quantitative and predictive understanding of nucleic acid folding.

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## Saturday, March 3, 2012

### Twin-core fiber optical tweezers: an optimum designation

Farzin Emami and Ammar Rahimi Kazerooni

Twin-core fiber optical tweezers (TCFOT) can be simulated using finite difference beam propagation method. We chose a tapered TCFOT and calculated its far-field pattern. It is found that, there is an optimum for the core index to have maximum peak at far-field pattern of a tapered TCFOT. This optimum value, can achieved a maximum force on the particles trapped at the far-field region of tapered TCFOT.

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### Optical trapping of metallic Rayleigh particle by combined beam

Ke Cheng, Xian-qiong Zhong and An-ping Xiang
Radiation forces and trapping stability of metallic (i.e. gold) Rayleigh particle by combined beam are analyzed, and the combined beam is formed by superimposing two partially coherent off-axis flat-topped beams. The dependences of radiation forces on off-axis distance parameter, correlation length and particle radius are illustrated by numerical examples. The results show that there exist critical values d 0,cand σ 0,c for the combined beam. For 0
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## Thursday, March 1, 2012

### Control of Plasmonic Superradiance in Metallic Nanoparticle Assembly by Light-Induced Force and Fluctuations

Takuya Iida

The possibility of simultaneous control of the configuration and optical functions of a metallic nanoparticle (NP) assembly by light-induced force (LIF) and thermal fluctuations has been demonstrated on the basis of self-consistent theory of LIF and nonequilibrium dynamics. It has been clarified that the NPs are arranged parallel to the polarization of the focused laser beam under the balance of LIF and the electrostatic repulsive force due to the ions on the surface of NPs. Particularly, in such a NP assembly consisting of high-density NPs, the light-scattering rate (radiative decay) of localized surface plasmon polaritons (LSPPs) can be drastically enhanced to be greater than 100 meV (10 times that of single NPs), and the spectral width is also greatly broadened due to the superradiance effect. The results will provide a foundation of the principles for designing a NP assembly with controllable light scattering for highly efficient broad-band light energy conversion devices.

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### Single-molecule imaging of DNA pairing by RecA reveals a three-dimensional homology search

Anthony L. Forget & Stephen C. Kowalczykowski

DNA breaks can be repaired with high fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA. After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing and exchange of DNA strands. Homologous pairing involves DNA sequence-specific target location by the RecA–ssDNA complex. Despite decades of study, the mechanism of this enigmatic search process remains unknown. RecA is a DNA-dependent ATPase, but ATP hydrolysis is not required for DNA pairing and strand exchange, eliminating active search processes. Using dual optical trapping to manipulate DNA, and single-molecule fluorescence microscopy to image DNA pairing, we demonstrate that both the three-dimensional conformational state of the dsDNA target and the length of the homologous RecA–ssDNA filament have important roles in the homology search. We discovered that as the end-to-end distance of the target dsDNA molecule is increased, constraining the available three-dimensional (3D) conformations of the molecule, the rate of homologous pairing decreases. Conversely, when the length of the ssDNA in the nucleoprotein filament is increased, homology is found faster. We propose a model for the DNA homology search process termed ‘intersegmental contact sampling’, in which the intrinsic multivalent nature of the RecA nucleoprotein filament is used to search DNA sequence space within 3D domains of DNA, exploiting multiple weak contacts to rapidly search for homology. Our findings highlight the importance of the 3D conformational dynamics of DNA, reveal a previously unknown facet of the homology search, and provide insight into the mechanism of DNA target location by this member of a universal family of proteins.

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