Trapped Polystyrene Bead Research Articles

Shear relaxation governs fusion dynamics of biomolecular condensates

Phase-separated biomolecular condensates must respond agilely to biochemical and environmental cues in performing their wide-ranging cellular functions, but our understanding of condensate dynamics is lagging. Ample evidence now indicates biomolecular condensates as viscoelastic fluids, where shear stress relaxes at a finite rate, not instantaneously as in viscous liquids. Yet the fusion dynamics of condensate droplets has only been modeled based on viscous liquids, with fusion time given by the viscocapillary ratio (viscosity over interfacial tension). Here we used optically trapped polystyrene beads to measure the viscous and elastic moduli and the interfacial tensions of four types of droplets. Our results challenge the viscocapillary model, and reveal that the relaxation of shear stress governs fusion dynamics. These findings likely have implications for other dynamic processes such as multiphase organization, assembly and disassembly, and aging.

Influence of static electric field on Raman polarizability of optically trapped polystyrene beads.

The influence of static electric field of about 4.9 kV/cm on optically trapped polystyrene bead has been studied using micro-Raman spectroscopy. This experimental method could explore the response of electronic polarizability of molecules to the static electric field by measuring the Raman intensity. It is observed that, the atomic electrons in polystyrene will polarize to the extent that they induce a Raman intensity enhancements by a factor ranging from 0.004 to 0.64 for various spectral bands of polystyrene when exposed to the electric field from ~4.90 to 4.96 kV/cm.

Depth-Dependent Optical Stiffness Toward Water-Air Interface

This research attempted to quantify the optical stiffness of trapped polystyrene bead toward water-air interface. The optical tweezers with 975 nm wavelength was used to optically trap a single 3 micron diameter of bead in a water-contained cell with air exposed top water surface. The optical stiffness was justified on effective radius (r*) of the bead lateral spatial distribution. The scattered light signals due to the trapped bead at different laser focus height from the bottom glass-water interface (less than 20 µm) and laser trapping powers (1.7 mW to 7.5 mW) were analyzed to investigate the r* profile. It was found that within our experimental condition, r* was independent of focus height at fixed power and exponentially decay with respect to laser power at fixed focus height.

Modeling and calibrating nonlinearity and crosstalk in back focal plane interferometry for three-dimensional position detection.

Back focal plane (BFP) interferometry is frequently used to detect the motion of a single laser trapped bead in a photonic force microscope (PFM) system. Whereas this method enables high-speed and high-resolution position measurement, its measurement range is limited by nonlinearity coupled with crosstalk in three-dimensional (3-D) measurement, and validation of its measurement accuracy is not trivial. This Letter presents an automated calibration system in conjunction with a 3-D quadratic model to render rapid and accurate calibration of the laser measurement system. An actively controlled three-axis laser steering system and a high-speed vision-based 3-D particle tracking system are integrated to the PFM system to enable rapid calibration. The 3-D quadratic model is utilized to correct for nonlinearity and crosstalk and, thus, extend the 3-D position detection volume of BFP interferometry. We experimentally demonstrated a 12-fold increase in detection volume when applying the method to track the motion of a 2.0μm laser trapped polystyrene bead.

Precise control and measurement of solid-liquid interfacial temperature and viscosity using dual-beam femtosecond optical tweezers in the condensed phase.

We present a novel method of microrheology based on femtosecond optical tweezers, which in turn enables us to directly measure and control in situ temperature at microscale volumes at the solid-liquid interface. A noninvasive pulsed 780 nm trapped bead spontaneously responds to changes in its environment induced by a co-propagating 1560 nm pulsed laser due to mutual energy transfer between the solvent molecules and the trapped bead. Strong absorption of the hydroxyl group by the 1560 nm laser creates local heating in individual and binary mixtures of water and alcohols. "Hot Brownian motion" of the trapped polystyrene bead is reflected in the corner frequency deduced from the power spectrum. Changes in corner frequency values enable us to calculate the viscosity as well as temperature at the solid-liquid interface. We show that these experimental results can also be theoretically ratified.

A clogging-free microfluidic platform with an incorporated pneumatically driven membrane-based active valve enabling specific membrane capacitance and cytoplasm conductivity characterization of single cells

This study reports a microfluidic platform for clogging-free electrical property analysis of single cells. A pneumatically driven membrane-based active valve was integrated in this platform to unblock clogging events of constriction microchannels where pneumatic pressures were used to tune the deformation of a polydimethylsiloxan (PDMS) membrane serving as one wall of the constriction microchannel. The proposed platform was first tested to unblock trapped polystyrene beads at the entrance of constriction microchannels and then the characterization of the cellular electrical properties of lung cancer cells was successfully demonstrated. Results showed that the measured cytoplasm conductivity (0.74±0.20S/m) and specific membrane capacitance (2.17±0.58μF/cm2) of cells were consistent with the results from the previous publications (0.73±0.17S/m, and 2.00±0.60μF/cm2, respectively). Overall, this study has presented a microfluidic platform for single cell analysis with an enhanced function for unblocking cell aggregates at the entrance of microchannels.

An optical tweezer-based study of antimicrobial activity of silver nanoparticles

Understanding and characterizing microbial activity reduction in the presence of antimicrobial agents can help in the design and manufacture of antimicrobial drugs. We demonstrate the use of an optical tweezer setup in recording the changes in bacterial activity with time, induced by the presence of foreign bodies in a bacterial suspension. This is achieved by monitoring the fluctuations of an optically trapped polystyrene bead immersed in it. Examining the changes in the fluctuation pattern of the bead with time provides an accurate characterization of the reduction in the microbial activity. Here, we report on the effect of addition of silver nanoparticles on bacterial cultures of Pseudomonas aeroginosa, Escherichia coli and Bacillus subtilis. We observe a decrease in the bacterial activity with time for the investigated bacterial samples. This method in our opinion, enables one to track changes in bacterial activity levels as a function of time of contact with the antibacterial agent with greater efficacy than traditional cell counting methods.

Single-Molecule Protein Unfolding and Translocation by the AAA+ Protease, ClpXP

All cells employ ATP-fueled AAA+ proteases for protein-quality control and regulation. In the ClpXP protease, the ring-shaped AAA+ molecular motor ClpX first recognizes and mechanically unfolds specific protein substrates, then translocates the denatured polypeptide through a central axial pore into the barrel-shaped peptidase ClpP for degradation. Although the mechanisms of ATP utilization and substrate degradation have been extensively studied in solution and recently at the single-molecule level[1, 2], the molecular details underlying mechanical substrate unfolding and stepping along the polypeptide track remain unexamined.Using a dual-laser optical trap, we probed how ClpXP unfolds and translocates a multi-domain protein substrate at the single-molecule level. In our assay, a ClpXP-substrate complex is tethered between two trapped polystyrene beads held in a dumbbell configuration [2]. Motility records of ClpXP along the polypeptide track show unique, fingerprint-like, substrate unfolding and translocation events. Following rapid and cooperative unfolding of individual domains, we find that ClpX translocates the polypeptide into ClpP, taking small steps of 5-8 amino acids. The nature of the polypeptide track affects ClpXP mechanochemical activity. ClpX step size does not depend on ClpP, though we observe substantial substrate refolding and slippage events when only ClpX is examined. Our results support a power-stroke model of denaturation in which successful unfolding requires mechanical pulling by the enzyme to coincide with transient stochastic protein destabilization.[1] Shin, Y., Davis, J.H., Brau, R.R., Martin, A., Kenniston, J.A., Baker T.A., Sauer, R.T., Lang, M.J. Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease. PNAS, 106, 19340 (2009)[2] Aubin-Tam, M. E., Olivares, A. O., Sauer, R. T., Baker, T. A., Lang, M. J. Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine. Cell, 145, 257-67 (2011)

Three-dimensional imaging and force characterization of multiple trapped particles in low NA counterpropagating optical traps

An experimental characterization of the three-dimensional (3D) position and force constants, acting on one or multiple trapped polystyrene beads in a weak counterpropagating beams geometry is reported. The 3D position of the trapped particles is tracked by imaging with two synchronized CMOS cameras from two orthogonal views and used to determine the stiffness along all three spatial directions through power spectrum analysis and the equipartition method. For the case of three trapped beads we measure the dependence of the force constants on the counterpropagating beams waist separation. The maximal transverse stiffnesses, is about 0.1 pN/µm per mW at a beam waist separation of 67 µm whereas the longitudinal stiffness is approximately 20 times lower. The experimental findings are in reasonable agreement with a recent physical-geometric optics calculation.

SECONDARY CONVERGENCE IN FEMTOSECOND LASER TRAPPING

When femtosecond laser pulses pass through a trapped polystyrene bead, water breakdown is induced even though the energy of laser pulse is much lower compared to the threshold value of breakdown when the femtosecond laser directly irradiates in water. This mechanism is assigned to the secondary convergence of the laser by the trapped bead.

Independent trapping, manipulation and characterization by an all-optical biophotonics workstation

Optical trapping has enabled a multitude of applications focusing, in particular, on non-invasive studies of cellular material. The full potential of optical trapping has, however, not yet been exploited due to restricted access to the trapped samples, caused by high numerical aperture objectives needed to focus the trapping laser beams. Here, we use our recently developed biophotonics workstation to overcome this limitation by introducing probing and spectroscopic characterization of optically trapped particles in a side-view geometry perpendicular to the trapping beams rather than in the traditional top-view geometry parallel to the trapping beams. Our method is illustrated by CARS and fluorescence spectroscopy of trapped polystyrene beads. The side-view geometry opens intriguing possibilities for accessing trapped particles with optical as well as other types of probe methods independent from the trapping process.

Optical tweezer micromanipulation of filamentous fungi

Optical tweezers have been little used in experimental studies on filamentous fungi. We have built a simple, compact, easy-to-use, safe and robust optical tweezer system that can be used with brightfield, phase contrast, differential interference contrast and fluorescence optics on a standard research grade light microscope. We have used this optical tweezer system in a range of cell biology applications to trap and micromanipulate whole fungal cells, organelles within cells, and beads. We have demonstrated how optical tweezers can be used to: unambiguously determine whether hyphae are actively homing towards each other; move the Spitzenkörper and change the pattern of hyphal morphogenesis; make piconewton force measurements; mechanically stimulate hyphal tips; and deliver chemicals to localized regions of hyphae. Significant novel experimental findings from our study were that germ tubes generated significantly smaller growth forces than leading hyphae, and that both hyphal types exhibited growth responses to mechanical stimulation with optically trapped polystyrene beads. Germinated spores that had been optically trapped for 25 min exhibited no deleterious effects with regard to conidial anastomosis tube growth, homing or fusion.

Probing collective dynamics of active particles using modulation force spectroscopy

In this letter, we report a method of measuring the dynamic viscosity of self-propelled active particles using an intensity-modulated optical tweezer. We have used a 6μm trapped polystyrene bead suspended in a bath of motile bacterial cells as a probe. The response function amplitude of the oscillatory bead directly measures the dynamics of the spatiotemporal structure of the motile particles. We find that unlike passive systems, the viscosity is defined by distributions of response function amplitudes that represent the long-range active temporal structures. Appropriate Langevin equations are set up that capture all these essential features.

Identification of biotic and abiotic particles by using a combination of optical tweezers and in situ Raman spectroscopy.

A highly versatile setup, which introduces an optical gradient trap into a Raman spectrometer, is presented. The particular configuration, which consists of two lasers, makes trapping independent from the Raman excitation laser and allows a separate adjustment of the trapping and excitation wavelengths. Thus, the excitation wavelength can be chosen according to the needs of the application. We describe the successful application of an optical gradient trap on transparent as well as on reflective, metal-coated microparticles. Raman spectra were recorded from optically trapped polystyrene beads and from single biological cells (e.g., erythrocytes, yeast cells). Also, metal-coated microparticles were trapped and used as surface enhanced Raman spectroscopy (SERS) substrates for tests on yeast cells. Furthermore, the optical gradient trap was combined with a SERS fiber probe. Raman spectra were recorded from trapped red blood cells using the SERS fiber probe for excitation.

Regulation of pseudopodia localization in lymphocytes through application of mechanical forces by optical tweezers.

T-lymphocytes are responsible for cell-mediated immunity, and recognize antigens on target cells (e.g., tumor cells, virus-infected cells) and antigen presenting cells (e.g., macrophages, dendritic cells). While mechanical forces applied to a cell surface can produce alterations in the cytoskeletal structure, leading to global structural rearrangements and changes in the intracellular biochemistry and gene expression, it remains unknown if local mechanical forces acting at the lymphocyte-antigen interaction site play any role in lymphocyte activation following antigen recognition. In this study we investigate the effect of such forces induced by optical tweezers on the lymphocyte's morphological response. We brought optically trapped polystyrene beads, coated with a specific antibody against a clonotypic epitope of the T-cell receptors (TCRs), in contact with individual lymphocytes and applied mechanical forces at the TCR-antibody interaction site. Although bead size was a factor, simple bead contact tended to induce formation of pseudopodia that appeared randomly over the cell's surface, while application of tangential forces at the interaction site redirected pseudopodia formation toward that site and promoted endocytosis activity. We propose that local forces play a key role in the initial lymphocyte adhesion to antigen-bearing cells, and may be implicated in antigen-specific motility, transendothelial migration, and tissue homing to sites of inflammation.

Effects of chelators (EGTA, EDTA) and calcium ions on nematode capture by the nematode-trapping fungus Arthrobotrys ellipsospora

The nematode-trapping fungusArthrobotrys ellipsospora developed an adhesive knob and trapped nematodes when cultured on a low-nutrient medium. It also trapped polystyrene beads in the same way. The adhesive knob produced mucus that was stained with alcian blue, while mycelium of the fungus was stained with periodic acid/Schiff (PAS). The amount of mucus increased with in days after culturing in the low-nutrient media. The fungus completely lost its ability to trap nematodes when treated with EDTA and EGTA, but it recovered the ability after incubation in the presence of a low concentration of Ca (10−6–10−7 M) for 1 h. Calmodulin inhibitor W-7 also inhibited the trapping ability of the fungus, and there was a significant (p<0.05) difference between the effects of W-7 and W-5. Ca-binding protein was also detected in the fungus.

Absence of a barrier to backwards rotation of the bacterial flagellar motor demonstrated with optical tweezers.

A cell of the bacterium Escherichia coli was tethered covalently to a glass coverslip by a single flagellum, and its rotation was stopped by using optical tweezers. The tweezers acted directly on the cell body or indirectly, via a trapped polystyrene bead. The torque generated by the flagellar motor was determined by measuring the displacement of the laser beam on a quadrant photodiode. The coverslip was mounted on a computer-controlled piezo-electric stage that moved the tether point in a circle around the center of the trap so that the speed of rotation of the motor could be varied. The motor generated approximately 4500 pN nm of torque at all angles, regardless of whether it was stalled, allowed to rotate very slowly forwards, or driven very slowly backwards. This argues against models of motor function in which rotation is tightly coupled to proton transit and back-transport of protons is severely limited.