Basudev Roy

Tumor-associated collagen signature-3 (TACS-3) is a prognostic indicator for breast cancer survival. It is characterized by highly organized, parallel bundles of collagen fibers oriented perpendicular to the tumor boundary, serving as directional, confining channels for cancer cell invasion. Here we design a TACS-3-mimetic anisotropic, confined collagen I matrix and examine the relation between anisotropy of matrix, directed cellular migration, and anisotropy of cell membrane-the first direct contact between TACS-3 and cell-using Michigan Cancer Foundation-7 (MCF-7) cells as cancer-model. Using unidirectional freezing, we generated ∼50 μm-wide channels filled with collagen I. Optical tweezer (OT) microrheology shows that anisotropic confinement increases collagen viscoelasticity by two orders of magnitude, and the elastic modulus is significantly greater along the direction of anisotropic confinement compared to that along the orthogonal direction, thus establishing matrix anisotropy. Furthermore, MCF-7 cells embedded in anisotropic collagen I, exhibit directionality in cellular morphology and migration. Finally, using customized OT to trap polystyrene probes bound to cell-membrane (and not to ECM) of either free cells or cells under anisotropic confinement, we quantified the effect of matrix anisotropy on membrane viscoelasticity, both in-plane and out-of-plane, vis-à-vis the membrane. Both bulk and viscous modulus of cell-membrane of MCF-7 cells exhibit significant anisotropy under anisotropic confinement. Moreover, the cell membrane of MCF-7 cells under anisotropic confinement is significantly softer (both in-plane and out-of-plane moduli) despite their local environment being five times stiffer than free cells. In order to test if the coupling between anisotropy of extracellular matrix and anisotropy of cell-membrane is regulated by cell-cytoskeleton, actin cytoskeleton was depolymerized for both free and confined cells. Results show that cell membrane viscoelasticity of confined MCF-7 cells is unaffected by actin de-polymerization, in contrast to free cells. Together, these findings suggest that anisotropy of ECM induces directed migration and correlates with anisotropy of cell-membrane viscoelasticity of the MCF-7 cells in an actin-independent manner.

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.

The fluctuation-dissipation relation, a central result in nonequilibrium statistical physics, relates equilibrium fluctuations in a system to its linear response to external forces. Here we provide a direct experimental verification of this relation for viscously coupled oscillators, as realized by a pair of optically trapped colloidal particles. A theoretical analysis, in which interactions mediated by slow viscous flow are represented by nonlocal friction tensors, matches experimental results and reveals a frequency maximum in the amplitude of the mutual response which is a sensitive function of the trap stiffnesses and the friction tensors. This allows for its location and width to be tuned and suggests the utility of the trap setup for accurate two-point microrheology.

3D Pitch rotational motion has been generated in spherical particles using holographic optical tweezers by maneuvering the laser spots to control the rotational motion. However, since the spherical particles, required to minimise complications due to the drag forces, are perfectly isotropic, a controllable torque cannot be applied with it. It is here that we trap birefringent particles in two tweezers beams and then change the depth of one of the beam foci controllably to generate a proper pitch rotational torque wrench. We also detect the rotation with our newly developed pitch rotational motion detection technique which could not be done conventionally on isotropic spherical particles.

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. This journal is

The cell membrane has fluctuations due to thermal and athermal sources. That causes the membrane to flicker. Conventionally, only the normal (perpendicular to the membrane) fluctuations are studied and then used to ascertain the membrane properties like the bending rigidity. It is here that we introduce a different concept, namely the slope fluctuations of the cell membrane which can be modelled as a gradient of the normal fluctuations. This can be studied using a new technique where a birefringent particle placed on the membrane turns in the out of plane sense, called the pitch sense. We introduce the pitch detection technique in optical tweezers relying upon asymmetric scattering from a birefringent particle under crossed polarizers. We then go on to use this pitch detection technique to ascertain the power spectral density of membrane slope fluctuations and find it to be (frequency)-1 while the normal fluctuations yields (frequency)-5/3. We also explore a different regime 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 now become (frequency)-4/3, the slope fluctuations spectrum still remains (frequency)-1, with exactly the same coefficient as the case when the drug was not applied. Thus, this presents a convenient opportunity to study the membrane parameters like bending rigidity as a function of time after applying the drug. This would be the first time the membrane bending rigidity could be studied as a function of time upon the application of Lat-B without reverting to AFM.

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.

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.

Optical tweezers are tools made of light that enable contactless pushing, trapping, and manipulation of objects, ranging from atoms to space light sails. Since the pioneering work by Arthur Ashkin in the 1970s, optical tweezers have evolved into sophisticated instruments and have been employed in a broad range of applications in the life sciences, physics, and engineering. These include accurate force and torque measurement at the femtonewton level, microrheology of complex fluids, single micro- and nano-particle spectroscopy, single-cell analysis, and statistical-physics experiments. This roadmap provides insights into current investigations involving optical forces and optical tweezers from their theoretical foundations to designs and setups. It also offers perspectives for applications to a wide range of research fields, from biophysics to space exploration.

A three dimensional rigid spherical microscopic object can rotate in either the pitch, yaw or roll fashion. Among these, yaw motion has been conventionally studied using the intensity of the scattered light from birefringent microspheres through crossed polarizers. So far, however, there is no way to study the pitch rotational motion in spherical microspheres. Here we suggest a new method towards the study of such pitch motion in birefringent microspheres under crossed polarizers by measuring the 2-fold asymmetry in the scattered signal using video microscopy. We show a simple example of pitch rotation determination using video microscopy for a microsphere attached with a kinesin molecule while moving along a microtubule. It can also be extended to optical tweezers.