Proper measurement of pure dielectrophoresis force acting on a RBC using optical tweezers.

The force experienced by a neutral dielectric object in the presence of a spatially non-uniform electric field is referred to as dielectrophoresis (DEP). The proper quantification of DEP force in the single-cell level could be of great importance for the design of high-efficiency micro-fluidic systems for the separation of biological cells. In this report we show how optical tweezers can be properly utilized for proper quantification of DEP force experienced by a human RBC. By tuning the temporal frequency of the applied electric field and also performing control experiments and comparing our experimental results with that of theoretically calculated, we show that the measured force is a pure DEP force. Our results show that in the frequency range of 0.1-3 M H z the DEP force acting on RBC is frequency independent.

Moiré deflectometry-based position detection for optical tweezers.

Optical tweezers have proven to be indispensable tools for pico-Newton range force spectroscopy. A quadrant photodiode (QPD) positioned at the back focal plane of an optical tweezers' condenser is commonly used for locating the trapped object. In this Letter, for the first time, to the best of our knowledge, we introduce a moiré pattern-based detection method for optical tweezers. We show, both theoretically and experimentally, that this detection method could provide considerably better position sensitivity compared to the commonly used detection systems. For instance, position sensitivity for a trapped 2.17 µm polystyrene bead is shown to be 71% better than the commonly used QPD-based detection method. Our theoretical and experimental results are in good agreement.

Digital holography based submicron thermometry.

Here we introduce a phase-shifting digital holography-based method to determine the temperature profile around an irradiated (sub-)micron spherical bead. The method utilizes a Mach-Zehnder interferometer implemented into an open setup microscope. The results of irradiated gold spheres with diameter of 400 nm and also silver-coated micron-sized silica beads embedded in silicone oil are presented. We show that the applied method is able to accurately determine the surface temperature with accuracy of 1 °C. Our experimental results perfectly confirm the theoretical prediction of temperature profile around the irradiated bead.

Dynamics of membrane nanotubes coated with I-BAR.

Membrane deformation is a necessary step in a number of cellular processes such as filopodia and invadopodia formation and has been shown to involve membrane shaping proteins containing membrane binding domains from the IRSp53-MIM protein family. In reconstituted membranes the membrane shaping domains can efficiently deform negatively charged membranes into tubules without any other proteins present. Here, we show that the IM domain (also called I-BAR domain) from the protein ABBA, forms semi-flexible nanotubes protruding into Giant Unilamellar lipid Vesicles (GUVs). By simultaneous quantification of tube intensity and tubular shape we find both the diameter and stiffness of the nanotubes. I-BAR decorated tubes were quantified to have a diameter of ~50 nm and exhibit no stiffening relative to protein free tubes of the same diameter. At high protein density the tubes are immobile whereas at lower density the tubes diffuse freely on the surface of the GUV. Bleaching experiments of the fluorescently tagged I-BAR confirmed that the mobility of the tubes correlates with the mobility of the I-BAR on the GUV membrane. Finally, at low density of I-BAR the protein upconcentrates within tubes protruding into the GUVs. This implies that I-BAR exhibits strong preference for negatively curved membranes.

Nanoscale phase behavior on flat and curved membranes.

The diverse physical properties of membranes play a critical role in many membrane associated biological processes. Proteins responsible for membrane transport can be affected by the lateral membrane order and lateral segregation of proteins is often controlled by the preference of certain membrane anchors for membrane phases having a physically ordered state. The dynamic properties of coexisting membrane phases are often studied by investigating their thermal behavior. Optical trapping of gold nanoparticles is a useful tool to generate local phase transitions in membranes. The high local temperatures surrounding an irradiated gold nanoparticle can be used to melt a part of a giant unilamellar lipid vesicle (GUV) which is then imaged using phase sensitive fluorophores embedded within the bilayer. By local melting of GUVs we reveal how a protein-free, one component lipid bilayer can mediate passive transport of fluorescent molecules by localized and transient pore formation. Also, we show how tubular membrane curvatures can be generated by optical pulling from the melted region on the GUV. This will allow us to measure the effect of membrane curvature on the phase transition temperature.

Crosstalk elimination in the detection of dual-beam optical tweezers by spatial filtering.

In dual-beam optical tweezers, the accuracy of position and force measurements is often compromised by crosstalk between the two detected signals, this crosstalk leading to systematic and significant errors on the measured forces and distances. This is true both for dual-beam optical traps where the splitting of the two traps is done by polarization optics and for dual optical traps constructed by other methods, e.g., holographic tweezers. If the two traps are orthogonally polarized, most often crosstalk is minimized by inserting polarization optics in front of the detector; however, this method is not perfect because of the de-polarization of the trapping beam introduced by the required high numerical aperture optics. Here we present a simple and easy-to-implement method to efficiently eliminate crosstalk. The method is based on spatial filtering by simply inserting a pinhole at the correct position and is highly compatible with standard back focal plane photodiode based detection of position and force. Our spatial filtering method reduces crosstalk up to five times better than polarization filtering alone. The effectiveness is dependent on pinhole size and distance between the traps and is here quantified experimentally and reproduced by theoretical modeling. The method here proposed will improve the accuracy of force-distance measurements, e.g., of single molecules, performed by dual-beam optical traps and hence give much more scientific value for the experimental efforts.

Sub-diffraction positioning of a two-photon excited and optically trapped quantum dot.

Colloidal quantum dots are luminescent long-lived probes that can be two-photon excited and manipulated by a single laser beam. Therefore, quantum dots can be used for simultaneous single molecule visualization and force manipulation using an infra-red laser. Here, we show that even a single optically trapped quantum dot, performing restricted Brownian motion within the focal volume, can be two-photon excited by the trapping laser beam and its luminescence can be detected by a camera. After two-photon excitation for a time long enough, the emitted light from the quantum dot is shown to blueshift. A quantum dot is much smaller than a diffraction limited laser focus and by mapping out the intensity of the focal volume and overlaying this with the positions visited by a quantum dot, a quantum dot is shown often to explore regions of the focal volume where the intensity is too low to render two-photon absorption likely. This is in accordance with the observation that a trapped quantum dot is only fluorescing 5-10 percent of the time. The results are important for realizing nano-scale quantum dot control and visualization and for correct interpretation of experiments using two-photon excited quantum dots as markers.

Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells.

With the success of in vitro single-molecule force measurements obtained in recent years, the next step is to perform quantitative force measurements inside a living cell. Optical traps have proven excellent tools for manipulation, also in vivo, where they can be essentially non-invasive under correct wavelength and exposure conditions. It is a pre-requisite for in vivo quantitative force measurements that a precise and reliable force calibration of the tweezers is performed. There are well-established calibration protocols in purely viscous environments; however, as the cellular cytoplasm is viscoelastic, it would be incorrect to use a calibration procedure relying on a viscous environment. Here we demonstrate a method to perform a correct force calibration inside a living cell. This method (theoretically proposed in Fischer and Berg-Sørensen (2007 J. Opt. A: Pure Appl. Opt. 9 S239)) takes into account the viscoelastic properties of the cytoplasm and relies on a combination of active and passive recordings of the motion of the cytoplasmic object of interest. The calibration procedure allows us to extract absolute values for the viscoelastic moduli of the living cell cytoplasm as well as the force constant describing the optical trap, thus paving the way for quantitative force measurements inside the living cell. Here, we determine both the spring constant of the optical trap and the elastic contribution from the cytoplasm, influencing the motion of naturally occurring tracer particles. The viscoelastic moduli that we find are of the same order of magnitude as moduli found in other cell types by alternative methods.

Axial potential mapping of optical tweezers for biopolymer stretching: the bead size matters.

Optical tweezers (OT) are widely used for pico (and femto)-Newton range force measurements. The appropriate choice of the bead size is not well understood for biopolymer stretching applications of OT. We have shown, both by theory and experiment, that wrong choice of the bead size could cause errors as large as 295% in the measured force. We provide a simple map for correct choice of the bead size and the direction of pulling for such applications. There is a good agreement between our theoretical and experimental results.

FBAR syndapin 1 recognizes and stabilizes highly curved tubular membranes in a concentration dependent manner.

Syndapin 1 FBAR, a member of the Bin-amphiphysin-Rvs (BAR) domain protein family, is known to induce membrane curvature and is an essential component in biological processes like endocytosis and formation and growth of neurites. We quantify the curvature sensing of FBAR on reconstituted porcine brain lipid vesicles and show that it senses membrane curvature at low density whereas it induces and reinforces tube stiffness at higher density. FBAR strongly up-concentrates on the high curvature tubes pulled out of Giant Unilamellar lipid Vesicles (GUVs), this sorting behavior is strongly amplified at low protein densities. Interestingly, FBAR from syndapin 1 has a large affinity for tubular membranes with curvatures larger than its own intrinsic concave curvature. Finally, we studied the effect of FBAR on membrane relaxation kinetics with high temporal resolution and found that the protein increases relaxation time of the tube holding force in a density-dependent fashion.

Polarization-induced stiffness asymmetry of optical tweezers.

A tightly focused, linearly polarized laser beam, so-called optical tweezers, is proven to be a useful micromanipulation tool. It is known that there is a stiffness asymmetry in the direction perpendicular to the optical axis inherited from the polarization state of the laser. In this Letter, we report our experimental results of stiffness asymmetry for different bead sizes measured at the optimal trapping condition. We also provide the results of our generalized Lorenz-Mie based calculations, which are in good agreement with our experimental results. We also compare our results with previous reports.

Role of condenser iris in optical tweezer detection system.

Optical tweezers have proven to be very useful in various scientific fields, from biology to nanotechnology. In this Letter we show, both by theory and experiment, that the interference intensity pattern at the back focal plane of the condenser consists of two distinguishable areas with anticorrelated intensity changes when the bead is moved in the axial direction. We show that the space angle defining the border of two areas linearly depends on the NA of the objective. We also propose a new octant photodiode, which could significantly improve the axial resolution compared to the commonly used quadrant photodiode technique.

The effect of immersion oil in optical tweezers.

Optimized optical tweezers are of great importance for biological micromanipulation. In this paper, we present a detailed electromagnetic-based calculation of the spatial intensity distribution for a laser beam focused through a high numerical aperture objective when there are several discontinuities in the optical pathway of the system. For a common case of 3 interfaces we have shown that 0.01 increase in the refractive index of the immersion medium would shift the optimal trapping depth by 3-4 µm (0.2-0.6 µm) for aqueous (air) medium. For the first time, We have shown that the alteration of the refractive index of the immersion medium can be also used in aerosol trapping provided that larger increase in the refractive index is considered.

Potential mapping of optical tweezers.

Optical tweezers are very often used for measurement of piconewton range forces. Depending on the displacement of the trapped bead, the trap may become stiffer which causes considerable underestimation of the measured force. We have shown, both by theory and experiment, that such a stiffening occurs for beads larger than 0.5 µm in radius. For the first time, we have shown that the displacement at which the stiffening starts is size dependent and that the stiffening starts at higher forces for larger beads. We have shown that for the applications, which simultaneous force measurement and position sensing are on demand (such as biopolymer stretching), mid-range sized (∼1.5 µm in radius) beads could be the best choice.

Phase contrast optical tweezers.

In this paper, for the first time, we report on systematic theoretical and experimental investigation of Phase Contrast Optical Tweezers (PCOT) which could be an indispensable tool for micromanipulation of the transparent micro and nano objects such as biological tissues and vesicles. The quadrant photodiode detection scheme and the power-spectrum calibration method is shown to be valid for this case. We have shown that the phase objective with new designed phase plates can provide nearly aberration-free condition at a desired depth. This could be a valuable advantage for simultaneous in-depth micro-manipulations and visualization of the sample.

Direct measurements of heating by electromagnetically trapped gold nanoparticles on supported lipid bilayers.

Absorption of electromagnetic irradiation results in significant heating of metallic nanoparticles, an effect which can be advantageously used in biomedical contexts. Also, metallic nanoparticles are presently finding widespread use as handles, contacts, or markers in nanometer scale systems, and for these purposes it is essential that the temperature increase associated with electromagnetic irradiation is not harmful to the environment. Regardless of whether the heating of metallic nanoparticles is desired or not, it is crucial for nanobio assays to know the exact temperature increase associated with electromagnetic irradiation of metallic nanoparticles. We performed direct measurements of the temperature surrounding single gold nanoparticles optically trapped on a lipid bilayer, a biologically relevant matrix. The lipid bilayer had incorporated fluorescent molecules which have a preference for either fluid or gel phases. The heating associated with electromagnetic radiation was measured by visualizing the melted footprint around the irradiated particle. The effect was measured for individual gold nanoparticles of a variety of sizes and for a variety of laser powers. The temperatures were highly dependent on particle size and laser power, with surface temperature increments ranging from a few to hundreds of degrees Celsius. Our results show that by a careful choice of gold nanoparticle size and strength of irradiating electromagnetic field, one can control the exact particle temperature. The method is easily applicable to any type of nanoparticle for which the photothermal effect is sought to be quantified.

Active-passive calibration of optical tweezers in viscoelastic media.

In order to use optical tweezers as a force measuring tool inside a viscoelastic medium such as the cytoplasm of a living cell, it is crucial to perform an exact force calibration within the complex medium. This is a nontrivial task, as many of the physical characteristics of the medium and probe, e.g., viscosity, elasticity, shape, and density, are often unknown. Here, we suggest how to calibrate single beam optical tweezers in a complex viscoelastic environment. At the same time, we determine viscoelastic characteristics such as friction retardation spectrum and elastic moduli of the medium. We apply and test a method suggested [M. Fischer and K. Berg-Sørensen, J. Opt. A, Pure Appl. Opt. 9, S239 (2007)], a method which combines passive and active measurements. The method is demonstrated in a simple viscous medium, water, and in a solution of entangled F-actin without cross-linkers.

Optimized optical trapping of gold nanoparticles.

Metallic nanoparticles are of significant interest due to their particular optical and biological applications. Gold nanoparticles are proven to be excellent candidate for in vivo micro-manipulation using Optical Tweezers. This manuscript reports on stable 3-D trapping of 9.5-254nm gold nanospheres using substantially decreased laser power. The lower limit is approximately 2 times smaller than previous record. 5.4nm gold nanospheres were trapped for only 2-3 seconds. For the first time, our experimental data verify the volume corrected Rayleigh model for particles smaller than 100nm in diameter. Measuring the maximum applicable force for gold nanoparticles, we have shown that a few tens of milli-Watts of laser power can produce pico-Newton range forces.

Optimizing immersion media refractive index improves optical trapping by compensating spherical aberrations.

The efficiency of an optical trap is limited by its axial strength. Light focused by oil-immersion objectives provides stronger traps but suffers from spherical aberrations, thus restricting the axial stability and working distance. By changing the refractive index of the immersion media we compensate spherical aberrations and measure axial trapping strengths at least twice as large as previously reported. Moreover, the spherical aberrations can be compensated at any desired depth. The improved trapping efficiency implies significantly less heating of the particles, thus diminishing previously published concerns about using gold nanoparticles as handles for optical manipulation.

Correlation between mechanical strength of messenger RNA pseudoknots and ribosomal frameshifting.

Programmed ribosomal frameshifting is often used by viral pathogens including HIV. Slippery sequences present in some mRNAs cause the ribosome to shift reading frame. The resulting protein is thus encoded by one reading frame upstream from the slippery sequence and by another reading frame downstream from the slippery sequence. Although the mechanism is not well understood, frameshifting is known to be stimulated by an mRNA structure such as a pseudoknot. Here, we show that the efficiency of frameshifting relates to the mechanical strength of the pseudoknot. Two pseudoknots derived from the Infectious Bronchitis Virus were used, differing by one base pair in the first stem. In Escherichia coli, these two pseudoknots caused frameshifting frequencies that differed by a factor of two. We used optical tweezers to unfold the pseudoknots. The pseudoknot giving rise to the highest degree of frameshifting required a nearly 2-fold larger unfolding force than the other. The observed energy difference cannot be accounted for by any existing model. We propose that the degree of ribosomal frameshifting is related to the mechanical strength of RNA pseudoknots. Our observations support the "9 A model" that predicts some physical barrier is needed to force the ribosome into the -1 frame. Also, our findings support the recent observation made by cryoelectron microscopy that mechanical interaction between a ribosome and a pseudoknot causes a deformation of the A-site tRNA. The result has implications for the understanding of genetic regulation, reading frame maintenance, tRNA movement, and unwinding of mRNA secondary structures by ribosomes.