Comparison of thermal and athermal dynamics of the cell membrane slope fluctuations in the presence and absence of Latrunculin-B.

Conventionally, only the normal cell membrane fluctuations have been studied and used to ascertain membrane properties like the bending rigidity. A new concept, the membrane local slope fluctuations was introduced recently (Vaippullyet al2020Soft Matter167606), which can be modelled as a gradient of the normal fluctuations. It has been found that the power spectral density (PSD) of slope fluctuations behave as (frequency)-1while the normal fluctuations yields (frequency)-5/3even on the apical cell membrane in the high frequency region. In this manuscript, we explore a different situation where the cell is applied with the drug Latrunculin-B which inhibits actin polymerization and find the effect on membrane fluctuations. We find that even as the normal fluctuations show a power law (frequency)-5/3as is the case for a free membrane, the slope fluctuations PSD remains (frequency)-1, with exactly the same coefficient as the case when the drug was not applied. Moreover, while sometimes, when the normal fluctuations at high frequency yield a power law of (frequency)-4/3, the pitch PSD still yields (frequency)-1. Thus, this presents a convenient opportunity to study membrane parameters like bending rigidity as a function of time after application of the drug, while the membrane softens. We also investigate the active athermal fluctuations of the membrane appearing in the PSD at low frequencies and find active timescales of slower than 1 s.

Comparison of translational and rotational modes towards passive rheology of the cytoplasm of MCF-7 cells using optical tweezers.

A colloidal particle placed inside the cell cytoplasm is enmeshed within a network of cytoskeletal fibres immersed in the cytosolic fluid. The translational mode is believed to yield different rheological parameters than the rotational mode, given that these modes stretch the fibers differently. We compare the parameters for Michigan Cancer Foundation-7 (MCF-7) cells in this manuscript and find that the results are well comparable to each other. At low values of 0 Hz viscosity, the rotational and translational viscoelasticity matches well. However, discrepancies appear at higher values which may indicate that the cytoskeletal modes involved in rotation and translation of the particle are getting invoked. We also show that the 0 Hz viscosity increases as the cell ages under the conditions of constant room temperature of 25°C on the sample chamber.

A hydro-thermophoretic trap for microparticles near a gold-coated substrate.

Optical tweezers have revolutionised micromanipulation from physics and biology to material science. However, the high laser power involved in optical trapping can damage biological samples. In this context, indirect trapping of microparticles and objects using fluid flow fields has assumed great importance. It has recently been shown that cells and particles can be turned in the pitch sense by opto-plasmonic heating of a gold surface constituting one side of a sample chamber. We extend that work to place two such hotspots in close proximity to each other to form a very unique configuration of flow fields forming an effective quasi-three-dimensional 'trap', assisted by thermophoresis. This is effectively a harmonic trap confining particles in all three dimensions without relying on other factors to confine the particles close to the surface. We use this to show indirect trapping of different types of upconverting particles and cells, and also show that we can approach a trap stiffness of 40 fN µm-1 indicating a weak confinement regime without relying on feedback.

Temporal evolution of viscoelasticity of soft colloid laden air-water interface: a multiple mode microrheology study.

Mechanical properties of particle laden interfaces is crucial for various applications. For water droplets containing soft microgel particles, passive microrheology studies have revealed that the dynamically varying surface area of the evaporating drop results in a viscous to viscoelastic transition along the plane of the interface. However, the behaviour of the medium orthogonal to the interface has been elusive to study using passive microrheology techniques. In this work, we employ optical tweezers and birefringent probe particles to extract the direction-resolved viscoelastic properties of the particle-laden interface. By using special types of birefringent tracer particles, we detect not only the in-plane translational mode but also the out-of-plane translational (perpendicular to the interface) and rotational modes. We first compare different passive methods of probing the viscoelasticity of the microgel laden interface of sessile drop and then study the modes perpendicular to the interface and the out-of-plane rotational mode using optical tweezers based passive microrheology. The viscoelasticity of the interface using two different methods, i.e., multiple-particle tracking passive microrheology using video microscopy and by trapping birefringent tracer particles in optical tweezers, relying on different models are studied and found to exhibit comparable trends. Interestingly, the mode orthogonal to the interface and the rotational mode also show the viscous to viscoelastic transition as the droplet evaporates, but with lesser viscoelasticity during the same evaporation time than the in-plane mode.

Calcium-channel-blockers exhibit divergent regulation of cancer extravasation through the mechanical properties of cancer cells and underlying vascular endothelial cells.

Cardiovascular and cancer illnesses often co-exist, share pathological pathways, and complicate therapy. In the context of the potential oncological role of cardiovascular-antihypertensive drugs (AHD), here we examine the role of calcium-channel blocking drugs on mechanics of extravasating cancer cells, choosing two clinically-approved calcium-channel blockers (CCB): Verapamil-hydrochloride and Nifedipine, as model AHD to simultaneously target cancer cells (MCF7 and or MDA231) and an underlying monolayer of endothelial cells (HUVEC). First, live-cell microscopy shows that exposure to Nifedipine increases the spreading-area, migration-distance, and frequency of transmigration of MCF-7 cells through the HUVEC monolayer, whereas Verapamil has the opposite effect. Next, impedance-spectroscopy shows that for monolayers of either endothelial or cancer cells, Nifedipine-treatment alone decreases the impedance of both cases, suggesting compromised cell-cell integrity. Furthermore, upon co-culturing MCF-7 on the HUVEC monolayers, Nifedipine-treated MCF-7 cells exhibit weaker impedance than Verapamil-treated MCF-7 cells. Following, fluorescent staining of CCB-treated cytoskeleton, focal adhesions, and cell-cell junction also indicated that Nifedipine treatment diminished the cell-cell integrity, whereas verapamil treatment preserved the integrity. Since CCBs regulate intracellular Ca2+, we next investigated if cancer cell's exposure to CCBs regulates calcium-dependent processes critical to extravasation, specifically traction and mechanics of plasma membrane. Towards this end, first, we quantified the 2D-cellular traction of cells in response to CCBs. Results show that exposure to F-actin depolymerizing drug decreases traction stress significantly only for Nifedipine-treated cells, suggesting an actin-independent mechanism of Verapamil activity. Next, using an optical tweezer to quantify the mechanics of plasma membrane (PM), we observe that under constant, externally-applied tensile strain, PM of Nifedipine-treated cells exhibits smaller relaxation-time than Verapamil and untreated cells. Finally, actin depolymerization significantly decreases MSD only for Verapamil treated cancer-cells and endothelial cells and not for Nifedipine-treated cells. Together, our results show that CCBs can have varied, mechanics-regulating effects on cancer-cell transmigration across endothelial monolayers. A judicious choice of CCBs is critical to minimizing the pro-metastatic effects of antihypertension therapy.

Realization of pitch-rotational torque wrench in two-beam optical tweezers.

3D Pitch (out-of-plane) rotational motion has been generated in spherical particles by maneuvering the laser spots of holographic optical tweezers. However, since the spherical particles, which are required to minimise drag are perfectly isotropic, a controllable torque cannot be applied with it. It remains free to spin about any axis even after moving the tweezers beams. It is here that we trap birefringent particles of about 3 µm diameter in two tweezers beams and then change the depth of one of the beam foci controllably to generate a pitch rotational torque-wrench and avoid the free spinning of the particle. We also detect the rotation with newly developed pitch motion detection technique and apply controlled torques on the particle.

Estimation of rolling work of adhesion at the nanoscale with soft probing using optical tweezers.

Conventionally, the work of adhesion at the nanoscale is estimated using an atomic force microscope with a tip of the size of 10 nm. It is pressed into a surface with nano-Newton forces and then retracted to ascertain when the tip breaks away from the surface. Thus this ensures "hard probing" of a surface. However there can be another configuration where the particle is barely placed into the surface when the work of adhesion attaches the particle to the surface and this can be called "soft probing". In this configuration, if a birefringent particle is confined in linearly polarized optical tweezers, and then the surface is moved in the direction tangential to the plane, a rolling motion can be induced. Study of this rolling motion can also show the work of adhesion. We use this configuration to find the rolling work of adhesion of a 3 µm diameter birefringent particle on a glass surface. We go on to study the effects of changing the surface to a hydrophobic slippery surface like polydimethyl siloxane (PDMS). Then we go on to show that even 500 nm diameter diamonds bearing nitrogen vacancy (NV) centers which are birefringent due to the stresses on the crystal could also be trapped and rolled to generate pitch (out-of-plane rotation) motion with 50 nm contact diameters. We find that this mode of soft probing yields a work of adhesion of about 1 mJ m-2 while the conventional nanoscale probing with atomic force microscopes (AFM) yields about 50 mJ m-2.

Breaking the diffraction limit in absorption spectroscopy using upconverting nanoparticles.

We employ a single optically trapped upconverting nanoparticle (UCNP) of NaYF4:Yb,Er of diameter about 100 nm as a subdiffractive source to perform absorption spectroscopy. The experimentally expected mode volume of 100 nm of the backscatter profile of the nanoparticle matches well with a numerical simulation of the dominant backscattering modes to confirm our assertion of achieving a source dimension considerably lower than the diffraction limit set by the excitation wavelength of 975 nm for the UCNP. We perform absorption spectroscopy of several diverse entities such as the dye Rhodamine B in water, a thin gold film of thickness 30 nm, and crystalline soft oxometalates micro-patterned on a glass substrate using the UCNP as a source. The initial results lead to unambiguous utility of UCNPs as single nanoscopic sources for absorption spectroscopy of ultra-small sample volumes (femtolitres), and lead us to hypothesize a possible Resonance Energy Transfer mechanism between the UCNP and the molecules of the ambient medium, which may even lead to single molecule absorption spectroscopy applications.

Pitch-rotational manipulation of single cells and particles using single-beam thermo-optical tweezers.

3D pitch rotation of microparticles and cells assumes importance in a wide variety of applications in biology, physics, chemistry and medicine. Applications such as cell imaging and injection benefit from pitch-rotational manipulation. Generation of such motion in single beam optical tweezers has remained elusive due to the complexities of generating high enough ellipticity perpendicular to the direction of propagation. Further, trapping a perfectly spherical object at two locations and subsequent pitch rotation hasn't yet been demonstrated to be possible. Here, we use hexagonal-shaped upconverting particles and single cells trapped close to a gold-coated glass cover slip in a sample chamber to generate complete 360 degree and continuous pitch motion even with a single optical tweezer beam. The tweezers beam passing through the gold surface is partially absorbed and generates a hot-spot to produce circulatory convective flows in the vicinity which rotates the objects. The rotation rate can be controlled by the intensity of the laser light. Thus such a simple configuration can turn the particle in the pitch sense. The circulatory flows in this technique have a diameter of about 5 µm which is smaller than those reported using acousto-fluidic techniques.

Detection of sub-degree angular fluctuations of the local cell membrane slope using optical tweezers.

Normal thermal fluctuations of the cell membrane have been studied extensively using high resolution microscopy and focused light, particularly at the peripheral regions of a cell. We use a single probe particle attached non-specifically to the cell-membrane to determine that the power spectral density is proportional to (frequency)-5/3 in the range of 5 Hz to 1 kHz. We also use a new technique to simultaneously ascertain the slope fluctuations of the membrane by relying upon the determination of pitch motion of the birefringent probe particle trapped in linearly polarized optical tweezers. In the process, we also develop the technique to identify pitch rotation to a high resolution using optical tweezers. We find that the power spectrum of slope fluctuations is proportional to (frequency)-1, which we also explain theoretically. We find that we can extract parameters like bending rigidity directly from the coefficient of the power spectrum particularly at high frequencies, instead of being convoluted with other parameters, thereby improving the accuracy of estimation. We anticipate this technique for determination of the pitch angle in spherical particles to high resolution as a starting point for many interesting studies using the optical tweezers.

Measurement of viscoelastic properties of the cellular cytoplasm using optically trapped Brownian probes.

Measurement of the viscoelastic properties of a cell using microscopic tracer particles has been complicated given that the medium viscosity is dependent upon the size of the measurement probe leading to reliability issues. Further, a technique for direct calibration of optically trapped particles in vivo has been elusive due to the frequency dependence and spatial inhomogeneity of the cytoplasmic viscosity, and the requirement of accurate knowledge of the medium refractive index. Here, we employ a recent extension of Jeffery's model of viscoelasticity in the microscopic domain to fit the passive motional power spectra of micrometer-sized optically trapped particles embedded in a viscoelastic medium. We find excellent agreement between the 0 Hz viscosity in MCF7 cells and the typical values of viscosity in literature, between 2 to 16 mPa sec expected for the typical concentration of proteins inside the cytoplasmic solvent. This bypasses the dependence on probe size by relying upon small thermal displacements. Our measurements of the relaxation time also match values reported with magnetic tweezers, at about 0.1 s. Finally, we calibrate the optical tweezers and demonstrate the efficacy of the technique to the study of in vivo translational motion.

Detection of self-generated nanowaves on the interface of an evaporating sessile water droplet.

Evaporating sessile droplets have been known to exhibit oscillations on the air-liquid interface. These are generally over millimeter scales. Using a novel approach, we are able to measure surface height changes of 500 nm amplitude using optical trapping of a set of microscopic particles at the interface, particularly when the vertical thickness of the droplet reduces to less than 50 µm. We find that at the later stages of the droplet evaporation, particularly when the convection currents become large, the top air-water interface starts to spontaneously oscillate vertically as a function of time in consistency with predictions. We also detect travelling wave trains moving in the azimuthal direction of the drop surface which are consistent with hydrothermal waves at a different combination of Reynolds, Prandtl and Evaporation numbers than previously observed. This is the first time that wave-trains have been observed in water, being extremely challenging to detect both interferometrically and with infra-red cameras. We also find that such waves apply a force parallel to the interface along the propagation direction.