Line optical tweezers as controllable micromachines: techniques and emerging trends.

In the past three decades, the technology of optical tweezers has made significant contributions in various scientific areas, including optics, photonics, and nanosciences. Breakthroughs include manipulating particles in both static and dynamic ways, particle sorting, and constructing controllable micromachines. Advances in shaping and controlling the laser beam profile enable control over the position and location of the trap, which has many possible applications. A line optical tweezer (LOT) can be created by rapidly moving a spot optical tweezer using a tool such as a galvanometer mirror or an acousto-optic modulator. By manipulating the intensity profile along the beam line to be asymmetric or non-uniform, the technique can be adapted to various specific applications. Among the many exciting applications of line optical tweezers, in this work, we discuss in detail applications of LOT, including probing colloidal interactions, transporting and sorting of colloidal microspheres, self-propelled motions, trapping anisotropic particles, exploring colloidal interactions at fluid-fluid interfaces, and building optical thermal ratchets. We further discuss prospective applications in each of these areas of soft matter, including polymeric and biological soft materials.

AutoSmarTrace: Automated chain tracing and flexibility analysis of biological filaments.

Single-molecule imaging is widely used to determine statistical distributions of molecular properties. One such characteristic is the bending flexibility of biological filaments, which can be parameterized via the persistence length. Quantitative extraction of persistence length from images of individual filaments requires both the ability to trace the backbone of the chains in the images and sufficient chain statistics to accurately assess the persistence length. Chain tracing can be a tedious task, performed manually or using algorithms that require user input and/or supervision. Such interventions have the potential to introduce user-dependent bias into the chain selection and tracing. Here, we introduce a fully automated algorithm for chain tracing and determination of persistence lengths. Dubbed "AutoSmarTrace," the algorithm is built off a neural network, trained via machine learning to identify filaments within images recorded using atomic force microscopy. We validate the performance of AutoSmarTrace on simulated images with widely varying levels of noise, demonstrating its ability to return persistence lengths in agreement with input simulation parameters. Persistence lengths returned from analysis of experimental images of collagen and DNA agree with previous values obtained from these images with different chain-tracing approaches. Although trained on atomic-force-microscopy-like images, the algorithm also shows promise to identify chains in other single-molecule imaging approaches, such as rotary-shadowing electron microscopy and fluorescence imaging.

Sequence-dependent mechanics of collagen reflect its structural and functional organization.

Extracellular matrix mechanics influence diverse cellular functions, yet surprisingly little is known about the mechanical properties of their constituent collagen proteins. In particular, network-forming collagen IV, an integral component of basement membranes, has been far less studied than fibril-forming collagens. A key feature of collagen IV is the presence of interruptions in the triple-helix-defining (Gly-X-Y) sequence along its collagenous domain. Here, we used atomic force microscopy to determine the impact of sequence heterogeneity on the local flexibility of collagen IV and of the fibril-forming collagen III. Our extracted flexibility profile of collagen IV reveals that it possesses highly heterogeneous mechanics, ranging from semiflexible regions as found for fibril-forming collagens to a lengthy region of high flexibility toward its N-terminus. A simple model in which flexibility is dictated only by the presence of interruptions fit the extracted profile reasonably well, providing insight into the alignment of chains and demonstrating that interruptions, particularly when coinciding in multiple chains, significantly enhance local flexibility. To a lesser extent, sequence variations within the triple helix lead to variable flexibility, as seen along the continuously triple-helical collagen III. We found this fibril-forming collagen to possess a high-flexibility region around its matrix-metalloprotease binding site, suggesting a unique mechanical fingerprint of this region that is key for matrix remodeling. Surprisingly, proline content did not correlate with local flexibility in either collagen type. We also found that physiologically relevant changes in pH and chloride concentration did not alter the flexibility of collagen IV, indicating such environmental changes are unlikely to control its compaction during secretion. Although extracellular chloride ions play a role in triggering collagen IV network formation, they do not appear to modulate the structure of its collagenous domain.

Substrate stiffness tunes the dynamics of polyvalent rolling motors.

Nature has evolved many mechanisms for achieving directed motion on the subcellular level. The burnt-bridges ratchet (BBR) is one mechanism used to achieve superdiffusive molecular motion over long distances through the successive cleavage of surface-bound energy-rich substrate sites. This mechanism has been associated with both nanoscale and microscale movement, with the latter accomplished through polyvalent interactions between a large hub (e.g. influenza virus) and substrate (e.g. cell surface receptors). Experimental successes in achieving superdiffusive motion by synthetic polyvalent BBRs have raised questions about the dynamics of their motility, including whether rolling or translation is better able to direct motion of microscale spherical hubs. Here we simulate the three-dimensional dynamics of a polyvalent sphere moving on and cleaving an elastic substrate. We find that substrate stiffness plays an important role in controlling both the motor's mode of motility and its directional persistence. As we tune lateral substrate stiffness from soft to stiff we find there exists an intermediate value that optimizes rolling behaviour. We also find that there is an optimal substrate stiffness for maximizing persistence length, while stiffness does not influence as strongly the superdiffusive dynamics of the particle. Lastly, we examine the effect of substrate density, and show that softer landscapes are better able to buffer against decreases in substrate occupancy, with the spherical motor maintaining superdiffusive motion more on softer landscapes than on stiff landscapes as occupancy drops. Our results highlight the importance of surface in controlling the motion of polyvalent BBRs.

A chloride ring is an ancient evolutionary innovation mediating the assembly of the collagen IV scaffold of basement membranes.

Collagen IV scaffold is a principal component of the basement membrane (BM), a specialized extracellular matrix that is essential for animal multicellularity and tissue evolution. Scaffold assembly begins with the trimerization of α-chains into protomers inside the cell, which then are secreted and undergo oligomerization outside the cell. For the ubiquitous scaffold composed of α1- and α2-chains, both intracellular and extracellular stages are mediated by the noncollagenous domain (NC1). The association of protomers is chloride-dependent, whereby chloride ions induce interactions of the protomers' trimeric NC1 domains leading to NC1 hexamer formation. Here, we investigated the mechanisms, kinetics, and functionality of the chloride ion-mediated protomer assembly by using a single-chain technology to produce a stable NC1 trimer comprising α1, α2, and α1 NC1 monomers. We observed that in the presence of chloride, the single-chain NC1-trimer self-assembles into a hexamer, for which the crystal structure was determined. We discovered that a chloride ring, comprising 12 ions, induces the assembly of and stabilizes the NC1 hexamer. Furthermore, we found that the chloride ring is evolutionarily conserved across all animals, first appearing in cnidarians. These findings reveal a fundamental role for the chloride ring in the assembly of collagen IV scaffolds of BMs, a critical event enabling tissue evolution and development. Moreover, the single-chain technology is foundational for generating trimeric NC1 domains of other α-chain compositions to investigate the α121, α345, and α565 collagen IV scaffolds and to develop therapies for managing Alport syndrome, Goodpasture's disease, and cancerous tumor growth.

Environmentally Controlled Curvature of Single Collagen Proteins.

The predominant structural protein in vertebrates is collagen, which plays a key role in extracellular matrix and connective tissue mechanics. Despite its prevalence and physical importance in biology, the mechanical properties of molecular collagen are far from established. The flexibility of its triple helix is unresolved, with descriptions from different experimental techniques ranging from flexible to semirigid. Furthermore, it is unknown how collagen type (homo- versus heterotrimeric) and source (tissue derived versus recombinant) influence flexibility. Using SmarTrace, a chain-tracing algorithm we devised, we performed statistical analysis of collagen conformations collected with atomic force microscopy to determine the protein's mechanical properties. Our results show that types I, II, and III collagens-the key fibrillar varieties-exhibit similar molecular flexibilities. However, collagen conformations are strongly modulated by salt, transitioning from compact to extended as KCl concentration increases in both neutral and acidic pH. Although analysis with a standard worm-like chain model suggests that the persistence length of collagen can attain a wide range of values within the literature range, closer inspection reveals that this modulation of collagen's conformational behavior is not due to changes in flexibility but rather arises from the induction of curvature (either intrinsic or induced by interactions with the mica surface). By modifying standard polymer theory to include innate curvature, we show that collagen behaves as an equilibrated curved worm-like chain in two dimensions. Analysis within the curved worm-like chain model shows that collagen's curvature depends strongly on pH and salt, whereas its persistence length does not. Thus, we find that triple-helical collagen is well described as semiflexible irrespective of source, type, pH, and salt environment. These results demonstrate that collagen is more flexible than its conventional description as a rigid rod, which may have implications for its cellular processing and secretion.

Single-Molecule Assay for Proteolytic Susceptibility: Force-Induced Collagen Destabilization.

Force plays a key role in regulating dynamics of biomolecular structure and interactions, yet techniques are lacking to manipulate and continuously read out this response with high throughput. We present an enzymatic assay for force-dependent accessibility of structure that makes use of a wireless mini-radio centrifuge force microscope to provide a real-time readout of kinetics. The microscope is designed for ease of use, fits in a standard centrifuge bucket, and offers high-throughput, video-rate readout of individual proteolytic cleavage events. Proteolysis measurements on thousands of tethered collagen molecules show a load-enhanced trypsin sensitivity, indicating destabilization of the triple helix.

Modified Pluronic F127 Surface for Bioconjugation and Blocking Nonspecific Adsorption of Microspheres and Biomacromolecules.

Many experiments and applications require the chemical coupling of target molecules to surfaces, during which the elimination of nonspecific interactions presents a difficult challenge. We report on a technologically accessible surface passivation and chemical conjugation method based on an NHS end-labeled F127 Pluronic block copolymer (F127-NHS). To quantify interactions between the F127-NHS surface and magnetic microspheres, we developed a simple assay: the microsphere adhesion by gravity, inversion, then counting, or "MAGIC" assay. To improve blocking of microspheres while maintaining the ability to chemically couple additional molecules, we implemented a pH-dependent two-step chemical modification process for amine microspheres. This process achieves an extremely high level of blocking nonspecific interactions (less than 2% nonspecific adhesion) for a variety of microsphere surface charges and chemical functionalities. We also demonstrate the ability to specifically tether magnetic microspheres to an F127-NHS surface, using single DNA molecules. Using the DNA microspheres, we establish the applicability of the surface for force spectroscopy (stable with an applied load >30 pN) via the massively parallel technique of centrifuge force microscopy. Finally, we demonstrate that the surface can be used in fluorescence studies with a fluorogenic peptide cleavage assay, with high levels of blocking achieved for both the fluorogenic peptide and trypsin. These results suggest applications including, but not limited to, single-molecule force spectroscopy and fluorescence, biosensors, medical implants, and anti-biofouling, which could make use of the F127-NHS surface.

Effects of finite and discrete sampling and blur on microrheology experiments.

The frequency-dependent viscous and elastic properties of fluids can be determined from measurements of the thermal fluctuations of a micron-sized particle trapped by optical tweezers. Finite bandwidth and other instrument limitations lead to systematic errors in measurement of the fluctuations. In this work, we numerically represented power spectra of bead position measurements as if collected by two different measurement devices: a quadrant photodiode, which measures the deflection of the trapping laser; and a high-speed camera, which images the trapped bead directly. We explored the effects of aliasing, camera blur, sampling frequency, and measurement time. By comparing the power spectrum, complex response function, and the complex shear modulus with the ideal values, we found that the viscous and elastic properties inferred from the data are affected by the instrument limitations of each device. We discuss how these systematic effects might affect experimental results from microrheology measurements and suggest approaches to reduce discrepancies.

Intact Telopeptides Enhance Interactions between Collagens.

Collagen is the fundamental structural component of a wide range of connective tissues and of the extracellular matrix. It undergoes self-assembly from individual triple-helical proteins into well-ordered fibrils, a process that is key to tissue development and homeostasis, and to processes such as wound healing. Nucleation of this assembly is known to be slowed considerably by pepsin removal of short nonhelical regions that flank collagen's triple helix, known as telopeptides. Using optical tweezers to perform microrheology measurements, we explored the changes in viscoelasticity of solutions of collagen with and without intact telopeptides. Our experiments reveal that intact telopeptides contribute a significant frequency-dependent enhancement of the complex shear modulus. An analytical model of polymers associating to establish chemical equilibrium among higher-order species shows trends in G' and G″ consistent with our experimental observations, including a concentration-dependent crossover in G″/c around 300 Hz. This work suggests that telopeptides facilitate transient intermolecular interactions between collagen proteins, even in the acidic conditions used here.

Development and characterization of a eukaryotic expression system for human type II procollagen.

BACKGROUND: Triple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required. RESULTS: Here, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils. CONCLUSIONS: Using a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.

Construction and characterization of kilobasepair densely labeled peptide-DNA.

Directed assembly of biocompatible materials benefits from modular building blocks in which structural organization is independent of introduced functional modifications. For soft materials, such modifications have been limited. Here, long DNA is successfully functionalized with dense decoration by peptides. Following introduction of alkyne-modified nucleotides into kilobasepair DNA, measurements of persistence length show that DNA mechanics are unaltered by the dense incorporation of alkynes (∼1 alkyne/2 bp) and after click-chemistry attachment of a tunable density of peptides. Proteolytic cleavage of densely tethered peptides (∼1 peptide/3 bp) demonstrates addressability of the functional groups, showing that this accessible approach to creating hybrid structures can maintain orthogonality between backbone mechanics and overlaid function. The synthesis and characterization of these hybrid constructs establishes the groundwork for their implementation in future applications, such as building blocks in modular approaches to a range of problems in synthetic biology.

Fluence-dependent degradation of fibrillar type I collagen by 222 nm far-UVC radiation.

For more than 100 years, germicidal lamps emitting 254 nm ultraviolet (UV) radiation have been used for drinking-water disinfection and surface sterilization. However, due to the carcinogenic nature of 254 nm UV, these lamps have been unable to be used for clinical procedures such as wound or surgical site sterilization. Recently, technical advances have facilitated a new generation of germicidal lamp whose emissions centre at 222 nm. These novel 222 nm lamps have commensurate antimicrobial properties to 254 nm lamps while producing few short- or long-term health effects in humans upon external skin exposure. However, to realize the full clinical potential of 222 nm UV, its safety upon internal tissue exposure must also be considered. Type I collagen is the most abundant structural protein in the body, where it self-assembles into fibrils which play a crucial role in connective tissue structure and function. In this work, we investigate the effect of 222 nm UV radiation on type I collagen fibrils in vitro. We show that collagen's response to irradiation with 222 nm UV is fluence-dependent, ranging from no detectable fibril damage at low fluences to complete fibril degradation and polypeptide chain scission at high fluences. However, we also show that fibril degradation is significantly attenuated by increasing collagen sample thickness. Given the low fluence threshold for bacterial inactivation and the macroscopic thickness of collagenous tissues in vivo, our results suggest a range of 222 nm UV fluences which may inactivate pathogenic bacteria without causing significant damage to fibrillar collagen. This presents an initial step toward the validation of 222 nm UV radiation for internal tissue disinfection.

Motility of an autonomous protein-based artificial motor that operates via a burnt-bridge principle.

Inspired by biology, great progress has been made in creating artificial molecular motors. However, the dream of harnessing proteins - the building blocks selected by nature - to design autonomous motors has so far remained elusive. Here we report the synthesis and characterization of the Lawnmower, an autonomous, protein-based artificial molecular motor comprised of a spherical hub decorated with proteases. Its "burnt-bridge" motion is directed by cleavage of a peptide lawn, promoting motion towards unvisited substrate. We find that Lawnmowers exhibit directional motion with average speeds of up to 80 nm/s, comparable to biological motors. By selectively patterning the peptide lawn on microfabricated tracks, we furthermore show that the Lawnmower is capable of track-guided motion. Our work opens an avenue towards nanotechnology applications of artificial protein motors.

Squaring the circle in peptide assembly: from fibers to discrete nanostructures by de novo design.

The design of bioinspired nanostructures and materials of defined size and shape is challenging as it pushes our understanding of biomolecular assembly to its limits. In such endeavors, DNA is the current building block of choice because of its predictable and programmable self-assembly. The use of peptide- and protein-based systems, however, has potential advantages due to their more-varied chemistries, structures and functions, and the prospects for recombinant production through gene synthesis and expression. Here, we present the design and characterization of two complementary peptides programmed to form a parallel heterodimeric coiled coil, which we use as the building blocks for larger, supramolecular assemblies. To achieve the latter, the two peptides are joined via peptidic linkers of variable lengths to produce a range of assemblies, from flexible fibers of indefinite length, through large colloidal-scale assemblies, down to closed and discrete nanoscale objects of defined stoichiometry. We posit that the different modes of assembly reflect the interplay between steric constraints imposed by short linkers and the bulk of the helices, and entropic factors that favor the formation of many smaller objects as the linker length is increased. This approach, and the resulting linear and proteinogenic polypeptides, represents a new route for constructing complex peptide-based assemblies and biomaterials.

Through the Eyes of Creators: Observing Artificial Molecular Motors.

Inspired by molecular motors in biology, there has been significant progress in building artificial molecular motors, using a number of quite distinct approaches. As the constructs become more sophisticated, there is also an increasing need to directly observe the motion of artificial motors at the nanoscale and to characterize their performance. Here, we review the most used methods that tackle those tasks. We aim to help experimentalists with an overview of the available tools used for different types of synthetic motors and to choose the method most suited for the size of a motor and the desired measurements, such as the generated force or distances in the moving system. Furthermore, for many envisioned applications of synthetic motors, it will be a requirement to guide and control directed motions. We therefore also provide a perspective on how motors can be observed on structures that allow for directional guidance, such as nanowires and microchannels. Thus, this Review facilitates the future research on synthetic molecular motors, where observations at a single-motor level and a detailed characterization of motion will promote applications.

Positional stability of holographic optical traps.

The potential of digital holography for complex manipulation of micron-sized particles with optical tweezers has been clearly demonstrated. By contrast, its use in quantitative experiments has been rather limited, partly due to fluctuations introduced by the spatial light modulator (SLM) that displays the kinoforms. This is an important issue when high temporal or spatial stability is a concern. We have investigated the performance of both an analog-addressed and a digitally-addressed SLM, measuring the phase fluctuations of the modulated beam and evaluating the resulting positional stability of a holographic trap. We show that, despite imparting a more unstable modulation to the wavefront, our digitally-addressed SLM generates optical traps in the sample plane stable enough for most applications. We further show that traps produced by the analog-addressed SLM exhibit a superior pointing stability, better than 1 nm, which is comparable to that of non-holographic tweezers. These results suggest a means to implement precision force measurement experiments with holographic optical tweezers (HOTs).

Multivalent Diffusive Transport.

We present here a model for multivalent diffusive transport whereby a central point-like hub is coupled to multiple feet, which bind to complementary sites on a two-dimensional landscape. The available number of binding interactions is dependent on the number of feet (multivalency) and on their allowed distance from the central hub (span). Using Monte Carlo simulations that implement the Gillespie algorithm, we simulate multivalent diffusive transport processes for 100 distinct walker designs. Informed by our simulation results, we derive an analytical expression for the diffusion coefficient of a general multivalent diffusive process as a function of multivalency, span, and dissociation constant Kd. Our findings can be used to guide the experimental design of multivalent transporters, in particular, providing insight into how to overcome trade-offs between diffusivity and processivity.

Power spectral analysis for optical trap stiffness calibration from high-speed camera position detection with limited bandwidth.

The use of camera imaging enables trap calibration for multiple particles simultaneously. For stiff traps, however, blur from image integration time affects the detected particle positions significantly. In this paper we use power spectral analysis to calibrate stiff optical traps, taking the effects of blur, aliasing and position detection error into account, as put forward by Wong and Halvorsen [Opt. Express 14, 12517 (2006)]. We find agreement with simultaneously obtained photodiode data and the expected relation of corner frequency fc with laser power, up to fc = 3.6 kHz for a Nyquist frequency of 1.25 kHz. Spectral analysis enables easy identification of the contribution of noise. We demonstrate the utility of our approach with simultaneous calibration of multiple holographic optical traps.

Optical Tweezers Approaches for Probing Multiscale Protein Mechanics and Assembly.

Multi-step assembly of individual protein building blocks is key to the formation of essential higher-order structures inside and outside of cells. Optical tweezers is a technique well suited to investigate the mechanics and dynamics of these structures at a variety of size scales. In this mini-review, we highlight experiments that have used optical tweezers to investigate protein assembly and mechanics, with a focus on the extracellular matrix protein collagen. These examples demonstrate how optical tweezers can be used to study mechanics across length scales, ranging from the single-molecule level to fibrils to protein networks. We discuss challenges in experimental design and interpretation, opportunities for integration with other experimental modalities, and applications of optical tweezers to current questions in protein mechanics and assembly.