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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.

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.

Liquid–liquid phase separation of multivalent intrinsically disordered protein–RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA–protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA–protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA–protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein–RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein−RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein−RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.

This book intends to describe and review the extension of optical trapping and manipulation approaches to new materials beyond the classical dielectric beads.

Holographic optical tweezers (HOT) is a programmable technique used for manipulation of microsized samples. In combination with computer-generation holography (CGH), a spatial light modulator reshapes the light distribution within the focal area of the optical tweezers. HOT can be used to realize real-time multiple-point manipulation in fluid, and this is useful in biological research. In this article, we summarize the HOT technique, discuss its recent developments, and present an overview of its biological applications.

Cellulose biosynthesis in sessile bacterial colonies originates in the membrane-integrated bacterial cellulose synthase (Bcs) AB complex. We utilize optical tweezers to measure single-strand cellulose biosynthesis by BcsAB from Rhodobacter sphaeroides. Synthesis depends on uridine diphosphate glucose, Mg2+, and cyclic diguanosine monophosphate, with the last displaying a retention time of ~80 min. Below a stall force of 12.7 pN, biosynthesis is relatively insensitive to force and proceeds at a rate of one glucose addition every 2.5 s at room temperature, increasing to two additions per second at 37°. At low forces, conformational hopping is observed. Single-strand cellulose stretching unveiled a persistence length of 6.2 nm, an axial stiffness of 40.7 pN, and an ability for complexes to maintain a tight grip, with forces nearing 100 pN. Stretching experiments exhibited hysteresis, suggesting that cellulose microstructure underpinning robust biofilms begins to form during synthesis. Cellohexaose spontaneously binds to nascent single cellulose strands, impacting polymer mechanical properties and increasing BcsAB activity.

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.

Red blood cells are amazingly deformable structures able to recover their initial shape even after large deformations as when passing through tight blood capillaries. The reason for this exceptional property is found in the composition of the membrane and the membrane-cytoskeleton interaction. We investigate the mechanics and the dynamics of RBCs by a unique noninvasive technique, using weak optical tweezers to measure membrane fluctuation amplitudes with μs temporal and sub nm spatial resolution. This enhanced edge detection method allows to span over >4 orders of magnitude in frequency. Hence, we can simultaneously measure red blood cell membrane mechanical properties such as bending modulus κ = 2.8 ± 0.3 x 10⁻¹⁹J = 67.6 ± 7.2 kBT, tension σ = 6.5 ± 2.1 x 10⁻⁷N/m, and an effective viscosity ηeff = 81 ± 3.7 x 10⁻³ Pa s that suggests unknown dissipative processes. We furthermore show that cell mechanics highly depends on the membrane-spectrin interaction mediated by the phosphorylation of the interconnection protein 4.1R. Inhibition and activation of this phosphorylation significantly affects tension and effective viscosity. Our results show that on short time scales (slower than 100 ms) the membrane fluctuates as in thermodynamic equilibrium. At time scales longer than 100 ms, the equilibrium description breaks down and fluctuation amplitudes are higher by 40% than predicted by the membrane equilibrium theory. Possible explanations for this discrepancy are influences of the spectrin that is not included in the membrane theory or nonequilibrium fluctuations that can be accounted for by defining a nonthermal effective energy of up to Eeff = 1.4 ± 0.1 kBT, that corresponds to an actively increased effective temperature.

In this paper we discuss the implementation of a dynamic holographic optical tweezers using a liquid crystal spatial light modulator (LCSLM) assembly. The LCSLM assembly can realize reconfigurable binary holograms and generate beams with user defined phase profiles. Depending on the nature of the liquid crystal molecule used, the LCSLM can provide a hologram update rate of 60 Hz to at least 1440 Hz. Thus the LCSLM can provide a trapping beam whose phase profile can be updated in real time. Such an optical tweezers therefore can be termed as dynamic holographic optical tweezers. In this paper we will show how the LCSLM assembly can facilitate various beam manipulation mechanisms which in turn can be utilized to estimate the optical force acting on the microscopic particle being trapped.

Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA. The DNA double helix consists of two strands coiled around each other. However, there are many instances when DNA must be separated into its individual strands—for example, when the DNA sequence needs to be copied. These single-stranded structures are highly prone to damage. For protection, the single-stranded DNA can wrap around single-stranded DNA binding (SSB) proteins, which also control how other maintenance proteins interact with the DNA. SSB proteins from the bacteria species Escherichia coli wrap single-stranded DNA into a variety of topologies known as binding modes. By using a technique that uses a laser to exert forces on an individual DNA molecule, Suksombat et al. unraveled DNA from a single SSB protein. This revealed that the unraveling occurs in a series of steps that correspond well to the known binding modes. These steps also provide the energies required to unravel the single-stranded DNA. Further experiments showed that SSBs can slide along DNA without having to change their binding mode. The unraveling and sliding mechanisms are likely to be used by other proteins to gain access to DNA coated with SSBs. The next step is to understand how SSBs interact with these other proteins, and how their various wrapping configurations affect this interaction.