In the past three decades, the ability to optically manipulate biomolecules has spurred a new era of medical and biophysical research. Optical tweezers (OT) have enabled experimenters to trap, sort, and probe cells, as well as discern the structural dynamics of proteins and nucleic acids at single molecule level. The steady improvement in OT’s resolving power has progressively pushed the envelope of their applications; there are, however, some inherent limitations that are prompting researchers to look for alternatives to the conventional techniques. To begin with, OT are restricted by their one-dimensional approach, which makes it difficult to conjure an exhaustive three-dimensional picture of biological systems. The high-intensity trapping laser can damage biological samples, a fact that restricts the feasibility of in vivo applications. Finally, direct manipulation of biological matter at nanometer scale remains a significant challenge for conventional OT. A significant amount of literature has been dedicated in the last 10 years to address the aforementioned shortcomings. Innovations in laser technology and advances in various other spheres of applied physics have been capitalized upon to evolve the next generation OT systems. In this review, we elucidate a few of these developments, with particular focus on their biological applications. The manipulation of nanoscopic objects has been achieved by means of plasmonic optical tweezers (POT), which utilize localized surface plasmons to generate optical traps with enhanced trapping potential, and photonic crystal optical tweezers (PhC OT), which attain the same goal by employing different photonic crystal geometries. Femtosecond optical tweezers (fs OT), constructed by replacing the continuous wave (cw) laser source with a femtosecond laser, promise to greatly reduce the damage to living samples. Finally, one way to transcend the one-dimensional nature of the data gained by OT is to couple them to the other large family of single molecule tools, i.e., fluorescence-based imaging techniques. We discuss the distinct advantages of the aforementioned techniques as well as the alternative experimental perspective they provide in comparison to conventional OT.

We present a method for determining the free energy of coexisting states from irreversible work measurements. Our approach is based on a fluctuation relation that is valid for dissipative transformations in partially equilibrated systems. To illustrate the validity and usefulness of the approach, we use optical tweezers to determine the free energy branches of the native and unfolded states of a two-state molecule as a function of the pulling control parameter. We determine, within $0.6k_{B}T$ accuracy, the transition point where the free energies of the native and the unfolded states are equal.

We analyze the dynamics of a two-dimensional system of interacting active dumbbells. We characterize the mean-square displacement, linear response function, and deviation from the equilibrium fluctuation-dissipation theorem as a function of activity strength, packing fraction, and temperature for parameters such that the system is in its homogeneous phase. While the diffusion constant in the last diffusive regime naturally increases with activity and decreases with packing fraction, we exhibit an intriguing nonmonotonic dependence on the activity of the ratio between the finite-density and the single-particle diffusion constants. At fixed packing fraction, the time-integrated linear response function depends nonmonotonically on activity strength. The effective temperature extracted from the ratio between the integrated linear response and the mean-square displacement in the last diffusive regime is always higher than the ambient temperature, increases with increasing activity, and, for small active force, monotonically increases with density while for sufficiently high activity it first increases and next decreases with the packing fraction. We ascribe this peculiar effect to the existence of finite-size clusters for sufficiently high activity and density at the fixed (low) temperatures at which we worked. The crossover occurs at lower activity or density the lower the external temperature. The finite-density effective temperature is higher (lower) than the single dumbbell one below (above) a crossover value of the Péclet number.

The hypothesis that phase transitions originate from some topological change of the critical level hypersurface of the potential energy receives direct evident support by our study of the Bishop-Peyrard model of DNA thermal denaturation.