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In the past decade, microsphere-assisted nanoscopy has been developed rapidly to overcome the diffraction limit. However, due to the limited size and high surface curvature of microspheres, the magnified imaging still suffers from problems like limited view scope, imaging distortion, and low contrast. In this paper, we specialize in the imaging mechanism of microspheres and find irradiance as the key factor for microsphere imaging quality. Utilizing a modified optical tweezer system, we achieve precise manipulation of microspheres and further propose a high-quality large-field magnified imaging scheme. The results show that the imaging area of 5 µm microspheres can reach 16×12 µm 2 with the minimum identifiable feature of 137 nm. This scheme provides a new solution for extending the measuring scope of microsphere-assisted nanoscope, and will certainly promote the application of this technology in practice.

Understanding the folding mechanism of knotted and slipknotted proteins has attracted considerable interest. Due to their topological complexity, knotted and slipknotted proteins are predicted to fold slowly and involve large topological barriers. Molecular dynamics simulation studies suggest that a slipknotted conformation can serve as an important intermediate to help greatly reduce the topological difficulty during the folding of some knotted proteins. Here we use a single molecule optical tweezers technique to directly probe the folding of a small slipknotted protein AFV3-109. We found that stretching AFV3-109 can lead to the untying of the slipknot and complete unfolding of AFV3-109. Upon relaxation, AFV3-109 can readily refold back to its native slipknot conformation with high fidelity when the stretching force is lower than 6 pN. The refolding of AFV3-109 occurs in a sharp two-state like transition. Our results indicate that, different from knotted proteins, the folding of a slipknotted protein like AFV3-109 can be fast, and may not necessarily involve a high topological barrier.

Microspheres can break the diffraction limit and magnify nano-structure imaging, and with its advantages of low cost and label-free operation, microsphere-assisted imaging has become an irreplaceable tool in the life sciences and for precision measurements. However, the tiny size and limited imaging field of traditional solid microspheres cause difficulties when imaging large sample areas. Alternatively, droplets have similar properties to those of microspheres, with large surface curvature and refractive-index difference from the surrounding environment, and they can also serve as lenses to focus light for observation and imaging. Previous work has shown that droplets with controllable size can be generated using an optical tweezer system and can be driven by optical traps to move precisely like solid microspheres. Here, a novel microdroplet-assisted imaging technology based on optical tweezers is proposed that better integrates the generation, manipulation, and utilization of droplets. ARTICLE HIGHLIGHTS: •Controllable droplet generation in an optical tweezer system by using photogenerated droplets. •The generated droplets can aid imaging in the same way as can solid microspheres but with larger field of view and higher imaging quality. •Droplets can be cleaned easily from flexible samples without causing mechanical damage. [ABSTRACT FROM AUTHOR]

We researched the mechanical unfolding of protein domains in monomeric protein NuG2 and the tandem polyproteins (NuG2)8 and (NuG2)16 using a dual-trap optical tweezers system. By stretching NuG2 and its polyproteins, (NuG2)8 and (NuG2)16 at the constant pulling speed of 500 nm sâˆ'1, we achieved the mechanical unfolding force of each domain in these proteins. Besides, we calculated the energy dissipation of NuG2, (NuG2)8 and (NuG2)16 by measuring the area enclosed by stretching and relaxation traces. Our results represent a key step towards engineering artificial polyproteins with controllable mechanical force and energy dissipation properties for force-buffering and energy dissipator applications. [ABSTRACT FROM AUTHOR]

The highly focused laser beam is capable of confining micro-sized particle in its focus. This is widely known as optical trapping. The Janus particle is composed of two hemispheres with different refractive indexes. In a linearly polarized optical trap, the Janus particle tends to align itself to an orientation where the interface of the two hemispheres is parallel to the laser propagation as well as the polarization direction. This enables a controllable approach that rotates the trapped particle with fine accuracy and could be used in partial measurement. However, due to the complexity of the interaction of the optical field and refractive index distribution, the trapping trajectory of the Janus particle in the linearly polarized optical trap is still uncovered. In this paper, we focus on the dynamic trapping process and the steady position and orientation of the Janus particle in the optical trap from both simulation and experimental aspects. The trapping process recorded by a high speed camera coincides with the simulation result calculated using the T-matrix model, which not only reveals the trapping trajectory, but also provides a practical simulation solution for more complicated structures and trapping motions. [ABSTRACT FROM AUTHOR]

Developing angular trapping methods, which enable optical tweezers to rotate a micronsized bead, is of great importance for studies of biomacromolecules in a wide range of torque‐generation processes. Here a novel controlled angular trapping method based on model composite Janus particles is reported, which consist of two hemispheres made of polystyrene and poly(methyl methacrylate). Through computational and experimental studies, the feasibility to control the rotation of a Janus particle in a linearly polarized laser trap is demonstrated. The results show that the Janus particle aligned its two hemispheres interface parallel to the laser propagation direction and polarization direction. The rotational state of the particle can be directly visualized by using a camera. The rotation of the Janus particle in the laser trap can be fully controlled in real time by controlling the laser polarization direction. The newly developed angular trapping technique has the great advantage of easy implementation and real‐time controllability. Considering the easy chemical preparation of Janus particles and implementation of the angular trapping, this novel method has the potential of becoming a general angular trapping method. It is anticipated that this new method will significantly broaden the availability of angular trapping in the biophysics community. [ABSTRACT FROM AUTHOR]

Single-molecule force spectroscopy (SMFS) has become a powerful tool in investigating the mechanical unfolding/folding of proteins at the single-molecule level. Polyproteins made of tandem identical repeats have been widely used in atomic force microscopy (AFM)-based SMFS studies, where polyproteins not only serve as fingerprints to identify single-molecule stretching events, but may also improve statistics of data collection. However, the inherent assumption of such experiments is that all the domains in the polyprotein are equivalent and one SMFS trajectory of stretching a polyprotein made of n domains is equivalent to n trajectories of stretching a single domain. Such an assumption has not been validated experimentally. Using a small protein NuG2 and its polyprotein (NuG2)4 as model systems, here we use optical trapping (OT) to directly validate this assumption. Our results show that OT experiments on NuG2 and (NuG2)4 lead to identical parameters describing the unfolding and folding kinetics of NuG2, demonstrating that indeed stretching a polyprotein of NuG2 is equivalent to stretching single NuG2 in force spectroscopy experiments and thus validating the use of polyproteins in SMFS experiments. [ABSTRACT FROM AUTHOR]

• A simple and easy axial calibration and displacement tacking method of optical trapped bead is proposed to any in-use optical tweezers without hardware update. • A flexible axial calibration method by dynamic force balance is proposed to keep the same sample between calibration and measurement. • 5 nm axial resolution and 500 nm range is easy achieved by imaging processing method. • The deviation of ~10 nm between two axial focus planes in dual-trap setup is first experimentally noticed and ~1 pn force error of force spectroscopy data is predicted by calculation. High-precision axial displacement tracking of trapped beads is an indispensable feature of optical tweezers in advanced single-molecule studies. Here, we demonstrate an alternative method that enables axial calibration and tracking to be carried out on the same sample to avoid unnecessary errors. This method works by applying a dynamic force balance on a bead trapped between a piezoelectrically driven glass slide and an optical trap; in this configuration, the bead can be stopped precisely in different positions and imaged by a camera. A simple gradient algorithm is used to process the images into calibration data. After optimization of the calibration method and samples, our method exhibited better than 5 nm experimental axial resolution, with a measurement range of +/-500 nm around the objective focus at video speed. Moreover, for the first time, the deviation of the focusing plane in dual-trap optical tweezers was measured. We confirmed the axial deviation between two optical traps in our setup to be ~10 nm, corresponding to a force spectroscopy gage error of ~1 pN. This approach offers a favorable solution for in-use setup updating, as it can be seamlessly integrated into any optical tweezers system without requiring new hardware updates. [ABSTRACT FROM AUTHOR]

In recent years, Janus microspheres technology has been widely applied and developed in various fields. However, existing studies often rely on electron microscopy images, treating Janus microspheres as spherical to observe their submicron structures. This approach falls short in meeting the demand for precise measurement of the specific position and orientation of microspheres at the single-molecule scale. Here, we propose a standardized processing procedure that integrates multiple image processing algorithms to measure the position of Janus microspheres through the image capture. Our approach enhances the two-dimensional planar positioning accuracy of non-spherical Janus microspheres in micro-nano optical measurement systems. Utilizing this method, we can conduct a further research on the structure and motion characteristics of the microsphere at the single-particle scale. This method has been validated on a transmission illumination optical tweezers platforms, successfully calibrating the central position of the optical trap. Our work offers a greater convenience for measuring non-spherical Janus microspheres in liquid environments. It also provides a fresh perspective for subsequent studies on the motion trajectories and force dynamics of heterogeneous microspheres, showcasing its broad potential for application. • A standardized image processing procedure is proposed. • 2D positioning accuracy for non-spherical Janus microspheres is improved. • Controllable rotation of Janus microspheres with varying sizes and ratios is measured. • Research lays groundwork for Janus microspheres in single-molecule applications. [ABSTRACT FROM AUTHOR]

Acquiring events massively from single-molecule force spectroscopy (SMFS) experiments, which is crucial for revealing important biophysical information, is usually not straightforward. A significant amount of human labor is usually required to identify events in the measured force spectrum during measuring or before performing further data analysis. This prevents the experiment from being done in a fully-automated manner or scaling with the throughput of the measuring setup. In this work, we attempt to tackle this problem with a deep learning approach. A deep neural network model is developed to infer the occurrence of the events using the data stream from the measuring setup. We demonstrated that the proposed method could achieve high accuracy with force spectrums of a variety of samples from both optical tweezers and AFMs by learning from user-given samples instead of complicated manual algorithm designing or parameter tuning. Furthermore, we found that the trained model can be used to perform event detection on datasets measured from a different optical tweezer setup, showing the potential of being leveraged in more complex deep learning schemes. • This work proposed a user-friendly approach to massively locate the events of interest from experimental SMFS data streams. • To our best knowledge, this work is the first attempt to adopt deep learning methods to locate events in SMFS experiments. • It is demonstrated that the proposed method could learn to locate events for a variety of samples from different instruments including optical tweezers and AFMs. • This method could help to achieve automated measuring on SMFS high-throughput platforms or other deep-learning based SMFS signal analysis. [ABSTRACT FROM AUTHOR]