Quantification of the interaction forces between dengue virus and dopamine type-2 receptor using optical tweezers.

BACKGROUND: Dengue virus (DENV) causes the most significant mosquito-borne viral disease with a wide spectrum of clinical manifestation, including neurological symptoms associated with lethal dengue diseases. Dopamine receptors are expressed in central nervous system, and dopamine antagonists have been reported to exhibit antiviral activity against DENV infection in vivo and in vitro. Although identification of host-cell receptor is critical to understand dengue neuropathogenesis and neurotropism, the involvement of dopamine receptors in DENV infection remains unclear. RESULTS: We exploited the sensitivity and precision of force spectroscopy to address whether dopamine type-2 receptors (D2R) directly interact with DENV particles at the first step of infection. Using optical tweezers, we quantified and characterized DENV binding to D2R expressed on Chinese hamster ovary (CHO) cells. Our finding suggested that the binding was D2R- and DENV-dependent, and that the binding force was in the range of 50-60 pN. We showed that dopamine antagonists prochlorperazine (PCZ) and trifluoperazine (TFP), previously reported to inhibit dengue infection, interrupt the DENV-D2R specific binding. CONCLUSIONS: This study demonstrates that D2R could specifically recognize DENV particles and function as an attachment factor on cell surfaces for DENV. We propose D2R as a host receptor for DENV and as a potential therapeutic target for anti-DENV drugs.

In-vitro blood purification using tiny pinch holographic optical tweezers based on deep learning.

In-vitro blood purification is essential to a wide range of medical treatments, requiring fine-grained analysis and precise separation of blood components. Despite existing methods that can extract specific components from blood by size or by magnetism, there is not yet a general approach to efficiently filter blood components on demand. In this work, we introduce the first programmable non-contact blood purification system for accurate blood component detection and extraction. To accurately identify different cells and artificial particles in the blood, we collected and annotated a new blood component object detection dataset and trained a collection of deep-learning-based object detectors upon it. To precisely capture and extract desired blood components, we fabricated a microfluidic chip and set up a customized holographic optical tweezer to trap and move cells/particles in the blood. Empirically, we demonstrate that our proposed system can perform real-time blood fractionation with high precision reaching up to 96.89%, as well as high efficiency. Its scalability and flexibility open new research directions in blood treatment.

All-Optical Trapping and Programmable Transport of Gold Nanorods with Simultaneous Orientation and Spinning Control.

Gold nanorods (GNRs) are of special interest in nanotechnology and biomedical applications due to their biocompatibility, anisotropic shape, enhanced surface area, and tunable optical properties. The use of GNRs, for example, as sensors and mechanical actuators, relies on the ability to remotely control their orientation as well as their translational and rotational motion, whether individually or in groups. Achieving such particle control by using optical tools is challenging and exceeds the capabilities of conventional laser tweezers. We present a tool that addresses this complex manipulation problem by using a curve-shaped laser trap, enabling the optical capture and programmable transport of single and multiple GNRs along any trajectory. This type of laser trap combines confinement and propulsion optical forces with optical torque to transport the GNRs while simultaneously controlling their rotation (spinning) and orientation. The proposed system facilitates the light-driven control of GNRs and the quantitative characterization of their motion dynamics including transport speed, spinning frequency, orientation, and confinement strength. We experimentally demonstrate that remote control of the GNRs can be achieved both near a substrate surface (2D trapping) and deep within the sample (3D all-optical trapping). The motion dynamics of two sets of off-resonant GNRs, possessing similar aspect ratios but different resonance wavelengths, are analyzed to highlight the role played by their optical and mechanical properties in the optical manipulation process. The experimental results are supported by a theoretical model describing the observed motion dynamics of the GNRs. This optical manipulation tool can significantly facilitate applications of light-driven nanorods.

Optical Tweezers Assembled Nanodiamond Quantum Sensors.

Here we show that gradient force optical tweezers can be used to mediate the self-assembly of nanodiamonds into superstructures, which can serve as optically trapped nanoscale quantum probes with superior magnetic resonance sensing capabilities. Enhanced fluorescence rates from nitrogen-vacancy NV- defect centers enable rapid acquisition of optically detected magnetic resonance (ODMR), and shape-induced forces can improve both positioning accuracy and orientation control. The use of confocal imaging can isolate the signal from individual nanodiamonds within the assembly, thereby retaining the desirable properties of a single crystal probe. The improvements afforded by the use nanodiamond assemblies has the potential to resolve dynamic changes through, for example, real-time monitoring of the ODMR contrast.

Direct measurement of self-diffusiophoretic force generated by active colloids of different patch coverage using optical tweezers.

HYPOTHESIS: Synthetic micro/nanomotors are gaining extensive attention for various biomedical applications (especially in drug delivery) due to their ability to mimic the motion of biological micro/nanoscale swimmers. The feasibility of these applications relies on tight control of propulsion speed, direction, and type of motion (translation, circular, etc.) along with the exerted self-propulsive force. We propose to exploit the variation of both self-propulsion speed and force of active colloids with different patch coverages (with and without supporting layer) for engineering diffusiophoretic micro/nanomotors. EXPERIMENTS: The microswimmers were designed at various patch coverages (10°, 30°, and 90°) with (Ti/Pt) and without (Pt) an adhesion layer for the catalytic patch through glancing angle metal deposition (GLAD) technique. Mean-square displacement (MSD) analysis was performed to obtain the self-propulsion parameters like speed and angular speed. Using optical tweezers (OT), the self-propulsive force was measured from the force power spectral density. FINDINGS: The findings of our experiments suggest the non-requirement of any adhesion layer preceding the catalyst deposition since the Pt 10° colloidal batch had the maximal self-propulsion speed (4.61±0.3µm/s) and force (345±57fN) for 5% w/v H2O2 fuel concentration. Moreover, the self-propulsion speed and force decreased with increasing patch size, contrary to theoretical estimates. Also, the self-propulsive force obtained from MSD is 2 to 4 times lower in magnitude than the OT based force values. We believe that the self-propelling motion of the micromotors is possibly hindered due to interactions with the surface of the quartz cuvette during the optical microscopic analysis. Further, the MSD is limited to the self-propulsive motion in two dimensions. On the other hand, OT based force measurement involve trapping the particles in the bulk of the solution entirely avoiding the particle-substrate interactions. Hence, OT based force measurements are better than the propulsion velocity based stokes drag force estimates. We believe that this study can lay the foundation in designing efficient micro/nanomotors for translational biomedical applications.

Amphibious Hybrid Laser Tweezers for Fluid and Solid Domains.

Optical trapping is a potent tool for achieving precise and noninvasive manipulation of small objects in a vacuum and liquids. However, due to the substantial disparity between optical forces and interfacial adhesion, target objects should be suspended in fluid environments, rendering solid contact surfaces a restricted area for conventional optical tweezers. In this work, by relying on a single continuous wave (CW) laser, we demonstrate an optical manipulation system applicable for both fluid and solid domains, namely, amphibious hybrid laser tweezers. The key to our system lies in modulating the intensity of the CW laser with duration shorter than the dynamic thermal equilibrium time within objects, wherein strong thermal gradient forces with ∼6 orders of magnitude higher than the forces in optical tweezers are produced, enabling moving and trapping micro/nano-objects on solid interfaces. Thereby, CW laser-based optical tweezers and pulsed laser-based photothermal shock tweezers are seamlessly fused with the advantages of cost-effectiveness and simplicity. Our concept breaks the stereotype that CW lasers are limited to generating tiny forces and instead achieve ultrawide force generation spanning from femto-newtons (10-15 N) to (10-6 N). Our work expands the horizon of optical manipulation by seamlessly bridging its applications in fluid and solid environments and holds promise for inspiring optical manipulation techniques to perform more challenging tasks, which may unearth application scenarios in diverse fields such as fundamental physical research, nanofabrication, micro/nanorobotics, biomedicine, and cytology.

Tunneling nanotubes mediate KRas transport: Inducing tumor heterogeneity and altering cellular membrane mechanical properties.

Mutation in oncogene KRas plays a crucial role in the occurrence and progression of numerous malignant tumors. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. We hypothesize that oncogene KRas mutations are intrinsic to alterations in cellular mechanics that promote malignant tumor generation and progression. Here, we demonstrate the use of optical tweezers coupled with a confocal fluorescence imaging system and gene interference technique to reveal that the mutant KRas protein can be transported between homogeneous and heterogeneous tumor cells by tunneling nanotubes (TNTs), resulting in a significant reduction of membrane tension and acceleration of membrane phospholipid flow in the recipient cells. Simultaneously, the changes in membrane mechanical properties of the tumor cells also enhance the metastatic and invasive ability of the tumors, which further contribute to the deterioration of the tumors. This finding helps to clarify the association between oncogene mutations and changes in the mechanical properties of tumor cells, which provides a theoretical basis for the development of cancer treatment strategies. STATEMENT OF SIGNIFICANCE: Here, we present a laser confocal fluorescence system integrated with optical tweezers to observe the transfer of mutant KRasG12D protein from mutant cells to wild-type cells through TNTs. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. Our results demonstrate a significant decrease in membrane tension and an increase in membrane phospholipid flow in recipient cells. These alterations in mechanical properties augment the migration and invasive capabilities of tumor cells, contributing to tumor malignancy. Our findings propose that cellular mechanical properties could serve as new markers for tumor development, and targeting membrane tension may hold potential as a therapeutic strategy.

BMP4 regulates asymmetric Pkd2 distribution in mouse nodal immotile cilia and ciliary mechanosensing required for left-right determination.

BACKGROUND: Mouse nodal immotile cilia mechanically sense the bending direction for left-right (L-R) determination and activate the left-side-specific signaling cascade, leading to increased Nodal activity. Asymmetric distribution of Pkd2, a crucial channel for L-R determination, on immotile cilia has been reported recently. However, the causal relationship between the asymmetric Pkd2 distribution and direction-dependent flow sensing is not well understood. Furthermore, the underlying molecular mechanism directing this asymmetric Pkd2 distribution remains unclear. RESULTS: The effects of several recombinant proteins and inhibitors on the Pkd2 distribution were analyzed using super-resolution microscopy. Notably, bone morphogenetic protein 4 (BMP4) affected the Pkd2 distribution. Additionally, three-dimensional manipulation of nodal immotile cilia using optical tweezers revealed that excess BMP4 caused defects in the mechanosensing ability of the cilia. CONCLUSIONS: Experimental data together with model calculations suggest that BMP4 regulates the asymmetric distribution of Pkd2 in nodal immotile cilia, thereby affecting the ability of these cilia to sense the bending direction for L-R determination. This study, for the first time, provides insight into the relationship between the asymmetric protein distribution in cilia and their function.

Swelling, Protein Adsorption, and Biocompatibility of Pectin-Chitosan Hydrogels.

The study aims to determine how chitosan impacts pectin hydrogel's ability to attach peritoneal leukocytes, activate complement, induce hemolysis, and adsorb blood proteins. The hydrogels PEC-Chi0, PEC-Chi25, PEC-Chi50, and PEC-Chi75 were prepared by placing a mixture solution of 4% pectin and 4% chitosan in a ratio of 4:0, 3:1, 2:2, and 1:3 in a solution of 1.0 M CaCl2. Chitosan was found to modify the mechanical properties of pectin-calcium hydrogels, such as hardness and cohesiveness-to-adhesiveness ratio. Chitosan in the pectin-calcium hydrogel caused pH-sensitive swelling in Hanks' solution. The PEC-Chi75 hydrogel was shown to adsorb serum proteins at pH 7.4 to a greater extent than other hydrogels. PEC-Chi75's strong adsorption capacity was related to lower peritoneal leukocyte adherence to its surface when compared to other hydrogels, showing improved biocompatibility. Using the optical tweezers approach, it was shown that the force of interaction between pectin-chitosan hydrogels and plasma proteins increased from 10 to 24 pN with increasing chitosan content from 0 to 75%. Thus, the properties of pectin-calcium hydrogel, which determine interactions with body tissues after implantation, are improved by the addition of chitosan, making pectin-chitosan hydrogel a promising candidate for smart biomaterial development.

Optical Halo: A Proof of Concept for a New Broadband Microrheology Tool.

Microrheology, the study of material flow at micron scales, has advanced significantly since Robert Brown's discovery of Brownian motion in 1827. Mason and Weitz's seminal work in 1995 established the foundation for microrheology techniques, enabling the measurement of viscoelastic properties of complex fluids using light-scattering particles. However, existing techniques face limitations in exploring very slow dynamics, crucial for understanding biological systems. Here, we present a proof of concept for a novel microrheology technique called "Optical Halo", which utilises a ring-shaped Bessel beam created by optical tweezers to overcome existing limitations. Through numerical simulations and theoretical analysis, we demonstrate the efficacy of the Optical Halo in probing viscoelastic properties across a wide frequency range, including low-frequency regimes inaccessible to conventional methods. This innovative approach holds promise for elucidating the mechanical behaviour of complex biological fluids.

Cationic Residues of the HIV-1 Nucleocapsid Protein Enable DNA Condensation to Maintain Viral Core Particle Stability during Reverse Transcription.

The HIV-1 nucleocapsid protein (NC) is a multifunctional viral protein necessary for HIV-1 replication. Recent studies have demonstrated that reverse transcription (RT) completes in the intact viral capsid, and the timing of RT and uncoating are correlated. How the small viral core stably contains the ~10 kbp double stranded (ds) DNA product of RT, and the role of NC in this process, are not well understood. We showed previously that NC binds and saturates dsDNA in a non-specific electrostatic binding mode that triggers uniform DNA self-attraction, condensing dsDNA into a tight globule against extending forces up to 10 pN. In this study, we use optical tweezers and atomic force microscopy to characterize the role of NC's basic residues in dsDNA condensation. Basic residue mutations of NC lead to defective interaction with the dsDNA substrate, with the constant force plateau condensation observed with wild-type (WT) NC missing or diminished. These results suggest that NC's high positive charge is essential to its dsDNA condensing activity, and electrostatic interactions involving NC's basic residues are responsible in large part for the conformation, size, and stability of the dsDNA-protein complex inside the viral core. We observe DNA re-solubilization and charge reversal in the presence of excess NC, consistent with the electrostatic nature of NC-induced DNA condensation. Previous studies of HIV-1 replication in the presence of the same cationic residue mutations in NC showed significant defects in both single- and multiple-round viral infectivity. Although NC participates in many stages of viral replication, our results are consistent with the hypothesis that cationic residue mutations inhibit genomic DNA condensation, resulting in increased premature capsid uncoating and contributing to viral replication defects.

Robotic Nanomanipulation Based on Spatiotemporal Modulation of Optical Gradients.

Robotic nanomanipulation emerges as a cutting-edge technique pivotal for in situ nanofabrication, advanced sensing, and comprehensive material characterization. In this study, we develop an optical robotic platform (ORP) for the dynamic manipulation of colloidal nanoparticles (NPs). The ORP incorporates a human-in-the-loop control mechanism enhanced by real-time visual feedback. This feature enables the generation of custom optical landscapes with adjustable intensity and phase configurations. Based on the ORP, we achieve the parallel and reconfigurable manipulation of multiple NPs. Through the application of spatiotemporal phase gradient-reversals, our platform demonstrates capabilities in trapping, binding, rotating, and transporting NPs across custom trajectories. This presents a previously unidentified paradigm in the realm of in situ nanomanipulation. Additionally, the ORP facilities a "capture-and-print" assembly process, utilizing a strategic interplay of phase and intensity gradients. This process operates under a constant laser power setting, streamlining the assembly of NPs into any targeted configuration. With its precise positioning and manipulation capabilities, underpinned by the spatiotemporal modulation of optical gradients, the ORP will facilitate the development of colloid-based sensors and on-demand fabrication of nanodevices.

Mechanical Characterization of the Erythrocyte Membrane Using a Capacitor-Based Technique.

Pathological processes often change the mechanical properties of cells. Increased rigidity could be a marker of cellular malfunction. Erythrocytes are a type of cell that deforms to squeeze through tiny capillaries; changes in their rigidity can dramatically affect their functionality. Furthermore, differences in the homeostatic elasticity of the cell can be used as a tool for diagnosis and even for choosing the adequate treatment for some illnesses. More accurate types of equipment needed to study biomechanical phenomena at the single-cell level are very costly and thus out of reach for many laboratories around the world. This study presents a simple and low-cost technique to study the rigidity of red blood cells (RBCs) through the application of electric fields in a hand-made microfluidic chamber that uses a capacitor principle. As RBCs are deformed with the application of voltage, cells are observed under a light microscope. From mechanical force vs. deformation data, the elastic constant of the cells is determined. The results obtained with the capacitor-based method were compared with those obtained using optical tweezers, finding good agreement. In addition, P. falciparum-infected erythrocytes were tested with the electric field applicator. Our technique provides a simple means of testing the mechanical properties of individual cells.

Controlled Second Harmonic Generation with Optically Trapped Lithium Niobate Nanoparticles.

We report the second harmonic generation (SHG) response from a single 34 nm diameter lithium niobate nanoparticle. The experimental setup involves a first beam devoted to the optical trapping of single nanoparticles, whereas a second arm involves a femtosecond laser source leading to the SHG emission from the trapped nanoparticles. SHG operation where one to three nanoparticles are present in the optical trap is first demonstrated, highlighting the transition between coherent and incoherent SHG, the latter known as hyper-Rayleigh scattering (HRS). With a spatial light modulator moving the optical trap in and out of the focus of the femtosecond beam, the SHG intensity is switched back and forth between a low and a high level. This controlled operation opens new avenues for nanoparticle characterization and applications in sensing or communication and information technologies and constitutes the first step in the design of active substrateless metasurfaces.

Life under tension: the relevance of force on biological polymers.

Optical tweezers have elucidated numerous biological processes, particularly by enabling the precise manipulation and measurement of tension. One question concerns the biological relevance of these experiments and the generalizability of these experiments to wider biological systems. Here, we categorize the applicability of the information garnered from optical tweezers in two distinct categories: the direct relevance of tension in biological systems, and what experiments under tension can tell us about biological systems, while these systems do not reach the same tension as the experiment, still, these artificial experimental systems reveal insights into the operations of biological machines and life processes.

Towards the understanding of molecular motors and its relationship with local unfolding.

Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding, catalysis, or release of products is coupled to the work performed by the molecular motor is still not entirely clear. This is due, in part, to a lack of understanding of the role of force in the mechanical-structural processes involved in enzyme catalysis. From a mechanical perspective, one promising hypothesis is the Haldane-Pauling hypothesis which considers the idea that part of the enzymatic catalysis is strain-induced. It suggests that enzymes cannot be efficient catalysts if they are fully complementary to the substrates. Instead, they must exert strain on the substrate upon binding, using enzyme-substrate energy interaction (binding energy) to accelerate the reaction rate. A novel idea suggests that during catalysis, significant strain energy is built up, which is then released by a local unfolding/refolding event known as 'cracking'. Recent evidence has also shown that in catalytic reactions involving conformational changes, part of the heat released results in a center-of-mass acceleration of the enzyme, raising the possibility that the heat released by the reaction itself could affect the enzyme's integrity. Thus, it has been suggested that this released heat could promote or be linked to the cracking seen in proteins such as adenylate kinase (AK). We propose that the energy released as a consequence of ligand binding/catalysis is associated with the local unfolding/refolding events (cracking), and that this energy is capable of driving the mechanical work.

Characterization of the interaction between the Sec61 translocon complex and ppαF using optical tweezers.

The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N-terminal signal peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction. However, a detailed biophysical characterization of this binding is still missing. Here, optical tweezers force spectroscopy was used to characterize the kinetic parameters of the dissociation process between Sec61 and the SP of prepro-alpha-factor. The unbinding parameters including off-rate constant and distance to the transition state were obtained by fitting rupture force data to Dudko-Hummer-Szabo models. Interestingly, the translocation inhibitor mycolactone increases the off-rate and accelerates the SP/Sec61 dissociation, while also weakening the interaction. Whereas the translocation deficient mutant containing a single point mutation in the SP abolished the specificity of the SP/Sec61 binding, resulting in an unstable interaction. In conclusion, we characterize quantitatively the dissociation process between the signal peptide and the translocon, and how the unbinding parameters are modified by a translocation inhibitor.

Dependence of nucleosome mechanical stability on DNA mismatches.

The organization of nucleosomes into chromatin and their accessibility are shaped by local DNA mechanics. Conversely, nucleosome positions shape genetic variations, which may originate from mismatches during replication and chemical modification of DNA. To investigate how DNA mismatches affect the mechanical stability and the exposure of nucleosomal DNA, we used an optical trap combined with single-molecule FRET and a single-molecule FRET cyclization assay. We found that a single base-pair C-C mismatch enhances DNA bendability and nucleosome mechanical stability for the 601-nucleosome positioning sequence. An increase in force required for DNA unwrapping from the histone core is observed for single base-pair C-C mismatches placed at three tested positions: at the inner turn, at the outer turn, or at the junction of the inner and outer turn of the nucleosome. The results support a model where nucleosomal DNA accessibility is reduced by mismatches, potentially explaining the preferred accumulation of single-nucleotide substitutions in the nucleosome core and serving as the source of genetic variation during evolution and cancer progression. Mechanical stability of an intact nucleosome, that is mismatch-free, is also dependent on the species as we find that yeast nucleosomes are mechanically less stable and more symmetrical in the outer turn unwrapping compared to Xenopus nucleosomes.

Probing the stability and interdomain interactions in the ABC transporter OpuA using single-molecule optical tweezers.

Transmembrane transporter proteins are essential for maintaining cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the wealth of detailed structural and functional data available for these systems, our understanding of their dynamics and, consequently, how they function is generally limited. We introduce an innovative approach that enables us to directly measure the dynamics and stability of interdomain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally relevant motions and interdomain interactions of individual transmembrane transporter proteins in real time in a lipid bilayer.

Extracellular domain 2 of TSPAN4 governs its functions.

Tetraspanin 4, a protein with four transmembrane helices and three connecting loops, senses membrane curvature and localizes to membrane tubes. This enrichment in tubular membranes enhances its diverse interactions. While the transmembrane part of the protein likely contributes to curvature sensitivity, the possible roles of the ectodomains in curvature sensitivity of tetraspanin 4 are still unknown. Here, using micropipette aspiration combined with confocal microscopy and optical tweezers, we show that the extracellular loop 2 contributes to the curvature sensitivity and curvature-induced interactions of tetraspanin 4. To this end, we created truncated tetraspanin 4 mutants by deleting each of the connecting loops. Subsequently, we pulled membrane tubes from giant plasma membrane vesicles containing tetraspanin 4-GFP or its mutants while maintaining controllable membrane tension and curvature. Among the mutations tested, the removal of the extracellular loop 2 had the most significant impact on both the curvature sensitivity and interactions of tetraspanin 4. Based on the results, we suggest that the extracellular loop 2 regulates the affinity of tetraspanin 4 towards curved membranes and affects its lateral interactions.