High-Performance Suspension Bead Sensor Based on Optical Tweezers and Immuno-Rolling Circle Amplification.

In recent years, optical tweezers have become an effective bioassay tool due to their unique advantages, especially in combination with suspension beads, which can be applied to develop a high-performance analysis platform capable of high-quality imaging and stable signal output. However, the optical tweezer-assisted bead analysis is still at the early stage, and further development of different favorable methods is in need. Herein, we have first developed the optical tweezer-assisted immuno-rolling circle amplification (immuno-RCA) on beads for protein detection. Prostate-specific antigen was selected as the model analyte, and the immunosandwich structure on beads was built by the high affinity of "antibody-antigen". The "protein-nucleic acid" signals were effectively converted through the covalent coupling procedure of antibodies and oligonucleotides, further initiating the RCA reaction to achieve signal amplification. The individual beads with the strong irregular Brownian motion in a fluid environment were eventually trapped by the optical tweezers to acquire the accurate and high-quality signal. Compared with the conventional immunoassay on beads, the sensitivity of the developed strategy was increased by 587 times with a limit of detection of 4.29 pg/mL (0.13 pM), as well as excellent specificity, stability, and reproducibility. This study developed the new optical tweezer-assisted beads imaging strategy for protein targets, which has great potential for being applied to clinical serology research and expands the application of optical tweezers in the bioassays.

A Handle-Free, All-Protein-Based Optical Tweezers Method to Probe Protein Folding-Unfolding Dynamics.

Optical tweezers (OT) have evolved into powerful single molecule force spectroscopy tools to investigate protein folding-unfolding dynamics. To stretch a protein of interest using OT, the protein must be flanked with two double stranded DNA (dsDNA) handles. However, coupling dsDNA handles to the protein is often of low yield, representing a bottleneck in OT experiments. Here, we report a handle-free, all-protein-based OT method for investigating protein folding/unfolding dynamics. In this new method, we employed disordered elastin-like polypeptides (ELPs) as a molecular linker and the mechanically stable cohesin-dockerin (Coh-Doc) pair as the prey-bait system to enable the efficient capture and stretching of individual protein molecules. This novel approach was validated by using model proteins NuG2 and RTX-v, yielding experimental results comparable to those obtained by using the dsDNA handle approach. This new method provides a streamlined and efficient OT approach to investigate the folding-unfolding dynamics of proteins at the single molecule level, thus expanding the toolbox of OT-based single molecule force spectroscopy.

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.

Droplet-based optical trapping for cell separation in mock forensic samples.

Optical tweezers have a wide range of uses for mechanical manipulation of objects in the microscopic range. This includes both living and static cells in a variety of biomedical and research applications. Single-focus optical tweezers, formed by focusing a laser beam through a high numerical aperture immersion objective, create a significant force, which enables controlled transport of a variety of different cell types and morphologies in three dimensions. Optical tweezers have been previously reported to capture and separate spermatozoa from a reconstituted simulated postcoital sample. We report herein the development of a simplified, more efficient cell transfer protocol that can separate and isolate both spermatozoa as well as leukocytes, with similar efficiencies as those previously reported. The new cell transfer method was used to separate sperm cells from a reconstituted mixture of spermatozoa and vaginal epithelial cells, with complete STR profiles developed from 50 cells with little evidence of contribution from the female contributor to the mixture. This modified protocol was then used to separate 21 samples of enriched leukocytes, with trapped cells ranging from 5 to 22 cells. Complete STR profiles were developed from as few as 10 leukocytes. Thus, with minimal sample preparation and a short trapping time, this method has the potential to provide an alternative to traditional differential extraction methods for separation of sperm:nonsperm mixtures while also providing versatility for separation of cells with differing morphologies.

Detection of nasopharyngeal cancer cells using the laser tweezer Raman spectroscopy technology.

Nasopharyngeal cancer (NPC), which arises from the nasopharyngeal epithelial lining, is one of the common malignant otorhinolaryngological tumors in China. Due to its insidious anatomical location and highly invasive and metastatic features, it is challenging to detect NPC at early stages. In this work, a rapid laser tweezer Raman spectroscopic (LTRS) system was built and used to trap and characterize single NPC cells. Using LTRS, high-quality Raman signals of the normal nasopharyngeal cell line (NP69) and NPC cells could be successfully obtained. By analysing the Raman peaks, some unique changes were found in components, such as DNA, amide I and amide III, in NPC cells compared with normal cells. In addition, we also used a multivariate statistical algorithm to establish a diagnostic model for identifying NPC cells with an accuracy of 90.0%. These results demonstrate that LTRS in combination with the multivariate statistical analysis is a convenient and high-efficiency cell identification technology, providing a novel and rapid methodology for NPC detection at the single cell level.

Multiple miRNA Detection through a Suspended Microbead Array Encoded by Triple-Color Upconversion Luminescent Nanotags via Bi-Beam Splitter Hybrid-Multitrap Optical Tweezers.

In recent years, optical tweezers have become a novel tool for biodetection, and to improve the inefficiency of a single trap, the development of multitraps is required. Herein, we constructed a set of hybrid multitrap optical tweezers with the balance of stability and flexibility by the combination of two different beam splitters, a diffraction optical element (DOE) and galvano mirrors (GMs), to capture polystyrene (PS) microbeads in aqueous solutions to create an 18-trap suspended array. A sandwich hybridization strategy of DNA-miRNA-DNA was adopted to detect three kinds of target miRNAs associated with triple negative breast cancer (TNBC), in which different upconversion nanoparticles (UCNPs) with red, green, and blue emissions were applied as luminescent tags to encode the carrier PS microbeads to further indicate the levels of the targets. With encoded luminescent microbeads imaged by a three-channel microscopic system, the biodetection displayed high sensitivity with low limits of detection (LODs) of 0.27, 0.32, and 0.33 fM and exceptional linear ranges of 0.5 fM to 1 nM, 0.7 fM to 1 nM, and 1 fM to 1 nM for miR-343-3p, miR-155, and miR-199a-5p, respectively. In addition, this bead-based assay method was demonstrated to have the potential for being applied in patients' serum by satisfactory standard addition recovery experiment results.

Applying Optical Tweezers with TIRF Microscopy to Quantify Physical Interactions Between Organelles in the Plant Endomembrane System.

Plant organelles are associated with each other through tethering proteins at membrane contact sites (MCS). Methods such as total internal reflection fluorescence (TIRF) optical tweezers allow us to probe organelle interactions in live plant cells. Optical tweezers (focused infrared laser beams) can trap organelles that have a different refractive index to their surrounding medium (cytosol), whilst TIRF allows us to simultaneously image behaviors of organelles in the thin region of cortical cytoplasm. However, few MCS tethering proteins have so far been identified and tested in a quantitative manner. Automated routines (such as setting trapping laser power and controlling the stage speed and distance) mean we can quantify organelle interactions in a repeatable and reproducible manner. Here we outline a series of protocols which describe laser calibrations required to collect robust data sets, generation of fluorescent plant material (Nicotiana tabacum, tobacco), how to set up an automated organelle trapping routine, and how to quantify organelle interactions (particularly organelle interactions with the endoplasmic reticulum). TIRF-optical tweezers enable quantitative testing of putative tethering proteins to reveal their role in plant organelle associations at MCS. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Microscope system set-up and stability Basic Protocol 2: Generation of transiently expressed fluorescent tobacco tissue by Agrobacterium-mediated infiltration Basic Protocol 3: Setting up an automated organelle trapping routine Basic Protocol 4: Quantifying organelle interactions.

Classification of formalin-fixed bladder cancer cells with laser tweezer Raman spectroscopy.

Bladder cancer is a common cancer that is relatively hard to detect at an early stage because of its non-obvious symptoms. It is known that bladder cells can be found in urine samples which potentially could be used for early detection of bladder cancer. Raman spectroscopy is a powerful non-invasive tool for accessing biochemical information of cells. Combined with laser tweezers, to allow isolation of single cells, Raman spectroscopy has been used to characterise a number of bladder cells that might be found in a urine sample. Using principal component-canonical variates analysis (PC-CVA) and k-fold validation, the results shows that the invasive bladder cancer cells can be identified with accuracy greater than 87%. This demonstrates the potential of developing an early detection method that identifies the invasive bladder cancer cells in urine samples.

In-vivo programmable acoustic manipulation of genetically engineered bacteria.

Acoustic tweezers can control target movement through the momentum interaction between an acoustic wave and an object. This technology has advantages over optical tweezers for in-vivo cell manipulation due to its high tissue penetrability and strong acoustic radiation force. However, normal cells are difficult to acoustically manipulate because of their small size and the similarity between their acoustic impedance and that of the medium. In this study, we use the heterologous expression of gene clusters to generate genetically engineered bacteria that can produce numerous sub-micron gas vesicles in the bacterial cytoplasm. We show that the presence of the gas vesicles significantly enhances the acoustic sensitivity of the engineering bacteria, which can be manipulated by ultrasound. We find that by employing phased-array-based acoustic tweezers, the engineering bacteria can be trapped into clusters and manipulated in vitro and in vivo via electronically steered acoustic beams, enabling the counter flow or on-demand flow of these bacteria in the vasculature of live mice. Furthermore, we demonstrate that the aggregation efficiency of engineering bacteria in a tumour is improved by utilizing this technology. This study provides a platform for the in-vivo manipulation of live cells, which will promote the progress of cell-based biomedical applications.

Laser tweezer Raman spectroscopy combined with deep neural networks for identification of liver cancer cells.

Rapid identification of cancer cells is crucial for clinical treatment guidance. Laser tweezer Raman spectroscopy (LTRS) that provides biochemical characteristics of cells can be used to identify cell phenotypes through classification models in a non-invasive and label-free manner. However, traditional classification methods require extensive reference databases and clinical experience, which is challenging when sampling at inaccessible locations. Here, we describe a classification method combing LTRS with deep neural network (DNN) for differential and discriminative analysis of multiple liver cancer (LC) cells. By using LTRS, we obtained high-quality single-cell Raman spectra of normal hepatocytes (HL-7702) and liver cancer cell lines (SMMC-7721, Hep3B, HepG2, SK-Hep1 and Huh7). The tentative assignment of Raman peaks indicated that arginine content was elevated and phenylalanine, glutathione and glutamate content was decreased in liver cancer cells. Subsequently, we randomly selected 300 spectra from each cell line for DNN model analysis, achieving a mean accuracy of 99.2%, a mean sensitivity of 99.2% and a mean specificity of 99.8% for the identification and classification of multiple LC cells and hepatocyte cells. These results demonstrate the combination of LTRS and DNN is a promising method for rapid and accurate cancer cell identification at single cell level.

Biological cell trapping and manipulation of a photonic nanojet by a specific microcone-shaped optical fiber tip.

Trapping and manipulating mesoscopic biological cells with high precision and flexibility are very important for numerous biomedical applications. In particular, a photonic nanojet based on a non-resonance focusing phenomenon can serve as a powerful tool for manipulating red blood cells and tumor cells in blood. In this study, we demonstrate an approach to trap and drive cells using a high-quality photonic nanojet which is produced by a specific microcone-shaped optical-fiber tip. The dynamic chemical etching method is used to fabricate optical-fiber probes with a microcone-shaped tip. Optical forces and potentials exerted on a red blood cell by a microcone-shaped fiber tips are analyzed based on finite-difference time-domain calculations. Optical trapping and driving experiments are done using breast cancer cells and red blood cells. Furthermore, a cell chain is formed by adjusting the magnitude of the optical force. The real-time backscattering intensities of multiple cells are detected, and highly sensitive trapping is achieved. This microcone-shaped optical fiber probe is potentially a powerful device for dynamic cell assembly, optical sorting, and the precise diagnosis of vascular diseases.

Studying Dynein Mechanochemistry with an Optical Trap.

Molecular motors generate force and mechanical work to perform some of the most energy-demanding cellular processes, such as whole cell motility and cell division. These motors experience resistance from the viscoelastic environment of the surrounding cytoplasm, and opposing forces that can originate from other motors bound to cytoskeleton. Optical trapping is the most widely used method to measure the force-generating and force-response characteristics of motor proteins. Here we present the methodologies of three different optical trapping assays we use to measure how forces originating from external factors affect the microtubule-detachment rate and velocity of dynein. We also briefly discuss the remaining challenges and future directions of optical trapping studies of dyneins and other microtubule-based motors.

Versatile, facile and low-cost single-cell isolation, culture and sequencing by optical tweezer-assisted pool-screening.

Real-time image-based sorting of target cells in a precisely indexed manner is desirable for sequencing or cultivating individual human or microbial cells directly from clinical or environmental samples; however, the versatility of existing methods is limited as they are usually not broadly applicable to all cell sizes. Here, an optical tweezer-assisted pool-screening and single-cell isolation (OPSI) system is established for precise, indexed isolation of individual bacterial, yeast or human-cancer cells. A controllable static flow field that acts as a cell pool is achieved in a microfluidics chip, to enable precise and ready screening of cells of 1 to 40 µm in size by bright-field, fluorescence, or Raman imaging. The target cell is then captured by a 1064 nm optical tweezer and deposited as one-cell-harboring nanoliter microdroplets in a one-cell-one-tube manner. For bacterial, yeast and human cells, OPSI achieves a >99.7% target-cell sorting purity and a 10-fold elevated speed of 10-20 cells per min. Moreover, OPSI-based one-cell RNA-seq of human cancer cells yields high quality and reproducible single-cell transcriptome profiles. The versatility, facileness, flexibility, modularized design, and low cost of OPSI suggest its broad applications for image-based sorting of target cells.

Acousto-holographic reconstruction of whole-cell stiffness maps.

Accurate assessment of cell stiffness distribution is essential due to the critical role of cell mechanobiology in regulation of vital cellular processes like proliferation, adhesion, migration, and motility. Stiffness provides critical information in understanding onset and progress of various diseases, including metastasis and differentiation of cancer. Atomic force microscopy and optical trapping set the gold standard in stiffness measurements. However, their widespread use has been hampered with long processing times, unreliable contact point determination, physical damage to cells, and unsuitability for multiple cell analysis. Here, we demonstrate a simple, fast, label-free, and high-resolution technique using acoustic stimulation and holographic imaging to reconstruct stiffness maps of single cells. We used this acousto-holographic method to determine stiffness maps of HCT116 and CTC-mimicking HCT116 cells and differentiate between them. Our system would enable widespread use of whole-cell stiffness measurements in clinical and research settings for cancer studies, disease modeling, drug testing, and diagnostics.

Structural region essential for amyloid fibril formation in cytochrome c elucidated by optical trapping.

Amyloid fibril formation of cytochrome c is spatially and temporally controlled with a combined method of disulfide bond cross-linking of cysteine-introduced variants and optical trapping, identifying that the structural change in the region containing Ala83 is essential for the amyloid fibril formation.

Tethering Complex Proteins and Protein Complexes for Optical Tweezers Experiments.

Tethering proteins to force probes, typically micrometer-sized beads, is a prerequisite for dissecting their properties with optical tweezers. DNA handles serve as spacers between the tethered protein of interest and the bead surface. Attachment sites of the DNA handles to both the surface of beads and to the protein of interest must be mechanically stable for optical tweezers experiments. The most prominent method for attaching DNA handles to proteins utilizes thiol chemistry, linking modified DNA to engineered cysteines in the target protein. This method, although experimentally straightforward, is impractical for the large number of proteins that endogenously contain multiple or essential cysteines at undesired positions. Here, we describe two alternative approaches that take advantage of genetically encoded tag sequences in the target protein. The first method uses the enzymes Sfp and BirA, and the second uses the more recently described SpyTag-SpyCatcher system. We outline the process of generating the DNA handles themselves, as well as how to make the DNA-protein chimeras for carrying out optical tweezers experiments. These methods have robustly worked for several diverse and complex proteins, including ones that are difficult to produce or purify, and for protein-containing complexes such as the ribosome. They will be useful in cases where chemistry-based approaches are impractical or not feasible.

Using Optical Tweezers to Monitor Allosteric Signals Through Changes in Folding Energy Landscapes.

Signaling proteins are composed of conserved protein interaction domains that serve as allosteric regulatory elements of enzymatic or binding activities. The ubiquitous, structurally conserved cyclic nucleotide binding (CNB) domain is found covalently linked to proteins with diverse folds that perform multiple biological functions. Given that the structures of cAMP-bound CNB domains in different proteins are very similar, it remains a challenge to determine how this domain allosterically regulates such diverse protein functions and folds. Instead of a structural perspective, we focus our attention on the energy landscapes underlying the CNB domains and their responses to cAMP binding. We show that optical tweezers is an ideal tool to investigate how cAMP binding coupled to interdomain interactions remodel the energy landscape of the regulatory subunit of protein kinase A (PKA), which harbors two CNB domains. We mechanically manipulate and probe the unfolding and refolding behavior of the CNB domains as isolated structures or selectively as part of the PKA regulatory subunit, and in the presence and absence of cAMP. Optical tweezers allows us to dissect the changes in the energy landscape associated with cAMP binding, and to examine the allosteric interdomain interactions triggered by the cyclic nucleotide. This single molecule approach can be used to study other modular, multidomain signaling proteins found in nature.

High-Speed Optical Traps Address Dynamics of Processive and Non-Processive Molecular Motors.

Interactions between biological molecules occur on very different time scales, from the minutes of strong protein-protein bonds, down to below the millisecond duration of rapid biomolecular interactions. Conformational changes occurring on sub-ms time scales and their mechanical force dependence underlie the functioning of enzymes (e.g., motor proteins) that are fundamental for life. However, such rapid interactions are beyond the temporal resolution of most single-molecule methods. We developed ultrafast force-clamp spectroscopy (UFFCS), a single-molecule technique based on laser tweezers that allows us to investigate early and very fast dynamics of a variety of enzymes and their regulation by mechanical load. The technique was developed to investigate the rapid interactions between skeletal muscle myosin and actin, and then applied to the study of different biological systems, from cardiac myosin to processive myosin V, microtubule-binding proteins, transcription factors, and mechanotransducer proteins. Here, we describe two different implementations of UFFCS instrumentation and protocols using either acousto- or electro-optic laser beam deflectors, and their application to the study of processive and non-processive motor proteins.

Microtubule Dumbbells to Assess the Effect of Force Geometry on Single Kinesin Motors.

The cytoskeletal motors myosin, kinesin, and dynein and their corresponding tracks, actin and microtubules, are force generating ATPases responsible for motility and morphological changes at the intracellular, cellular, and tissue levels. The pioneering application of optical tweezers to measure the force-producing properties of cytoskeletal motors has provided an unparalleled understanding of their mechanochemistry. The mechanosensitivity of processive, microtubule-based motors has largely been studied in the optical trap using the "single-bead" assay, where a bead-attached motor is held adjacent to a cytoskeletal filament as it processively steps along it. However, because of the geometrical constraints in the conventional single-bead assay, the motor-filament bond is not only loaded parallel to the long axis of the filament, but also perpendicular to the long axis of the filament. This perpendicular force, which is inherent in the conventional single-bead assay, accelerates the motor-filament detachment and has not been carefully considered in prior experiments. An alternative approach is the "three-bead" assay, which was developed for the study of non-processive myosin motors. The vertical force component is minimized in this assay, and the total opposing force is mainly parallel to the microtubule. Experiments with kinesin show that microtubule attachment durations can be highly variable and last for up to tenfold longer times in the three-bead assay, compared to the single-bead assay. Thus, the ability of kinesin to bear mechanical load and remain attached to microtubules depends on the forces in more than one dimension. In this chapter, we provide detailed methods for preparing the proteins, buffers, flow chambers, and bead-filament assemblies for performing the three-bead assay with microtubules and their motors.

Measuring αß T-Cell Receptor-Mediated Mechanosensing Using Optical Tweezers Combined with Fluorescence Imaging.

T-cell antigen receptors (TCRs) are mechanosensors, which initiate a signaling cascade upon ligand recognition resulting in T-cell differentiation, homeostasis, effector and regulatory functions. An optical trap combined with fluorescence permits direct monitoring of T-cell triggering in response to force application at various concentrations of peptide-bound major histocompatibility complex molecules (pMHC). The technique mimics physiological shear forces applied as cells crawl across antigen-presenting surfaces during immune surveillance. True single molecule studies performed on single cells profile force-bond lifetime, typically seen as a catch bond, and conformational change at the TCR-pMHC bond on the surface of the cell upon force loading. Together, activation and single molecule single cell studies provide chemical and physical triggering thresholds as well as insight into catch bond formation and quaternary structural changes of single TCRs. The present methods detail assay design, preparation, and execution, as well as data analysis. These methods may be applied to a wide range of pMHC-TCR interactions and have potential for adaptation to other receptor-ligand systems.