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.

Combined Mutual Learning Net for Raman Spectral Microbial Strain Identification.

Infectious diseases pose a significant threat to global health, yet traditional microbiological identification methods suffer from drawbacks, such as high costs and long processing times. Raman spectroscopy, a label-free and noninvasive technique, provides rich chemical information and has tremendous potential in fast microbial diagnoses. Here, we propose a novel Combined Mutual Learning Net that precisely identifies microbial subspecies. It demonstrated an average identification accuracy of 87.96% in an open-access data set with thirty microbial strains, representing a 5.76% improvement. 50% of the microbial subspecies accuracies were elevated by 1% to 46%, especially for E. coli 2 improved from 31% to 77%. Furthermore, it achieved a remarkable subspecies accuracy of 92.4% in the custom-built fiber-optical tweezers Raman spectroscopy system, which collects Raman spectra at a single-cell level. This advancement demonstrates the effectiveness of this method in microbial subspecies identification, offering a promising solution for microbiology diagnosis.

Light Control in Microbial Systems.

Light is a key environmental component influencing many biological processes, particularly in prokaryotes such as archaea and bacteria. Light control techniques have revolutionized precise manipulation at molecular and cellular levels in recent years. Bacteria, with adaptability and genetic tractability, are promising candidates for light control studies. This review investigates the mechanisms underlying light activation in bacteria and discusses recent advancements focusing on light control methods and techniques for controlling bacteria. We delve into the mechanisms by which bacteria sense and transduce light signals, including engineered photoreceptors and light-sensitive actuators, and various strategies employed to modulate gene expression, protein function, and bacterial motility. Furthermore, we highlight recent developments in light-integrated methods of controlling microbial responses, such as upconversion nanoparticles and optical tweezers, which can enhance the spatial and temporal control of bacteria and open new horizons for biomedical applications.

Label-Free Techniques for Probing Biomolecular Condensates.

Biomolecular condensates play important roles in a wide array of fundamental biological processes, such as cellular compartmentalization, cellular regulation, and other biochemical reactions. Since their discovery and first observations, an extensive and expansive library of tools has been developed to investigate various aspects and properties, encompassing structural and compositional information, material properties, and their evolution throughout the life cycle from formation to eventual dissolution. This Review presents an overview of the expanded set of tools and methods that researchers use to probe the properties of biomolecular condensates across diverse scales of length, concentration, stiffness, and time. In particular, we review recent years' exciting development of label-free techniques and methodologies. We broadly organize the set of tools into 3 categories: (1) imaging-based techniques, such as transmitted-light microscopy (TLM) and Brillouin microscopy (BM), (2) force spectroscopy techniques, such as atomic force microscopy (AFM) and the optical tweezer (OT), and (3) microfluidic platforms and emerging technologies. We point out the tools' key opportunities, challenges, and future perspectives and analyze their correlative potential as well as compatibility with other techniques. Additionally, we review emerging techniques, namely, differential dynamic microscopy (DDM) and interferometric scattering microscopy (iSCAT), that have huge potential for future applications in studying biomolecular condensates. Finally, we highlight how some of these techniques can be translated for diagnostics and therapy purposes. We hope this Review serves as a useful guide for new researchers in this field and aids in advancing the development of new biophysical tools to study biomolecular condensates.

Correlating fluorescence microscopy, optical and magnetic tweezers to study single chiral biopolymers such as DNA.

Biopolymer topology is critical for determining interactions inside cell environments, exemplified by DNA where its response to mechanical perturbation is as important as biochemical properties to its cellular roles. The dynamic structures of chiral biopolymers exhibit complex dependence with extension and torsion, however the physical mechanisms underpinning the emergence of structural motifs upon physiological twisting and stretching are poorly understood due to technological limitations in correlating force, torque and spatial localization information. We present COMBI-Tweez (Combined Optical and Magnetic BIomolecule TWEEZers), a transformative tool that overcomes these challenges by integrating optical trapping, time-resolved electromagnetic tweezers, and fluorescence microscopy, demonstrated on single DNA molecules, that can controllably form and visualise higher order structural motifs including plectonemes. This technology combined with cutting-edge MD simulations provides quantitative insight into complex dynamic structures relevant to DNA cellular processes and can be adapted to study a range of filamentous biopolymers.

C-terminal Domain of T4 gene 32 Protein Enables Rapid Filament Reorganization and Dissociation.

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein essential for DNA replication. gp32 forms stable protein filaments on ssDNA through cooperative interactions between its core and N-terminal domain. gp32's C-terminal domain (CTD) is believed to primarily help coordinate DNA replication via direct interactions with constituents of the replisome. However, the exact mechanisms of these interactions are not known, and it is unclear how tightly-bound gp32 filaments are readily displaced from ssDNA as required for genomic processing. Here, we utilized truncated gp32 variants to demonstrate a key role of the CTD in regulating gp32 dissociation. Using optical tweezers, we probed the binding and dissociation dynamics of CTD-truncated gp32, *I, to an 8.1 knt ssDNA molecule and compared these measurements with those for full-length gp32. The *I-ssDNA helical filament becomes progressively unwound with increased protein concentration but remains significantly more stable than that of full-length, wild-type gp32. Protein oversaturation, concomitant with filament unwinding, facilitates rapid dissociation of full-length gp32 from across the entire ssDNA segment. In contrast, *I primarily unbinds slowly from only the ends of the cooperative clusters, regardless of the protein density and degree of DNA unwinding. Our results suggest that the CTD may constrain the relative twist angle of proteins within the ssDNA filament such that upon critical unwinding the cooperative interprotein interactions largely vanish, facilitating prompt removal of gp32. We propose a model of CTD-mediated gp32 displacement via internal restructuring of its filament, providing a mechanism for rapid ssDNA clearing during genomic processing.

The application of single-molecule optical tweezers to study disease-related structural dynamics in RNA.

RNA, a dynamic and flexible molecule with intricate three-dimensional structures, has myriad functions in disease development. Traditional methods, such as X-ray crystallography and nuclear magnetic resonance, face limitations in capturing real-time, single-molecule dynamics crucial for understanding RNA function. This review explores the transformative potential of single-molecule force spectroscopy using optical tweezers, showcasing its capability to directly probe time-dependent structural rearrangements of individual RNA molecules. Optical tweezers offer versatility in exploring diverse conditions, with the potential to provide insights into how environmental changes, ligands and RNA-binding proteins impact RNA behaviour. By enabling real-time observations of large-scale structural dynamics, optical tweezers emerge as an invaluable tool for advancing our comprehension of RNA structure and function. Here, we showcase their application in elucidating the dynamics of RNA elements in virology, such as the pseudoknot governing ribosomal frameshifting in SARS-CoV-2.

Horizontal magnetic tweezers and its applications in single molecule micromanipulation experiments.

Magnetic tweezers (MTs) have become indispensable tools for gaining mechanistic insights into the behavior of DNA-processing enzymes and acquiring detailed, high-resolution data on the mechanical properties of DNA. Currently, MTs have two distinct designs: vertical and horizontal (or transverse) configurations. While the vertical design and its applications have been extensively documented, there is a noticeable gap in comprehensive information pertaining to the design details, experimental procedures, and types of studies conducted with horizontal MTs. This article aims to address this gap by providing a concise overview of the fundamental principles underlying transverse MTs. It will explore the multifaceted applications of this technique as an exceptional instrument for scrutinizing DNA and its interactions with DNA-binding proteins at the single-molecule level.

Integrating magnetic tweezers and single-molecule FRET: A comprehensive approach to studying local and global conformational changes simultaneously.

This chapter presents the integration of magnetic tweezers with single-molecule FRET technology, a significant advancement in the study of nucleic acids and other biological systems. We detail the technical aspects, challenges, and current status of this hybrid technique, which combines the global manipulation and observation capabilities of magnetic tweezers with the local conformational detection of smFRET. This innovative approach enhances our ability to analyze and understand the molecular mechanics of biological systems. The chapter serves as our first formal documentation of this method, offering insights and methodologies developed in our laboratory over the past decade.

Magnetic tweezers principles and promises.

Magnetic tweezers have become popular with the outbreak of single molecule micromanipulation: catching a single molecule of DNA, RNA or a single protein and applying mechanical constrains using micron-size magnetic beads and magnets turn out to be easy. Various factors have made this possible: the fact that manufacturers have been preparing these beads to catch various biological entities-the ease of use provided by magnets which apply a force or a torque at a distance thus inside a flow cell-some chance: since the forces so generated are in the right range to stretch a single molecule. This is a little less true for torque. Finally, one feature which also appears very important is the simplicity of their calibration using Brownian motion. Here we start by describing magnetic tweezers used routinely in our laboratory where we have tried to develop a device as simple as possible so that the experimentalist can really focus on the biological aspect of the biomolecules that he/she is interested in. We discuss the implications of the various components and their important features. Next, we summarize what is easy to achieve and what is less easy. Then we refer to contributions by other groups who have brought valuable insights to improve magnetic tweezers.

Construction and operation of high-resolution magnetic tape head tweezers for measuring single-protein dynamics under force.

Mechanical forces are critical to protein function across many biological contexts-from bacterial adhesion to muscle mechanics and mechanotransduction processes. Hence, understanding how mechanical forces govern protein activity has developed into a central scientific question. In this context, single-molecule magnetic tweezers has recently emerged as a valuable experimental tool, offering the capability to measure single proteins over physiologically relevant forces and timescales. In this chapter, we present a detailed protocol for the assembly and operation of our magnetic tape head tweezers instrument, specifically tailored to investigate protein dynamics. Our instrument boasts a simplified microscope design and incorporates a magnetic tape head as the force-generating apparatus, facilitating precise force control and enhancing its temporal stability, enabling the study of single protein mechanics over extended timescales spanning several hours or even days. Moreover, its straightforward and cost-effective design ensures its accessibility to the wider scientific community. We anticipate that this technique will attract widespread interest within the growing field of mechanobiology and expect that this chapter will provide facilitated accessibility to this technology.

Localized Plasmonic Heating for Single-Molecule DNA Rupture Measurements in Optical Tweezers.

To date, studies on the thermodynamic and kinetic processes that underlie biological function and nanomachine actuation in biological- and biology-inspired molecular constructs have primarily focused on photothermal heating of ensemble systems, highlighting the need for probes that are localized within the molecular construct and capable of resolving single-molecule response. Here we present an experimental demonstration of wavelength-selective, localized heating at the single-molecule level using the surface plasmon resonance of a 15 nm gold nanoparticle (AuNP). Our approach is compatible with force-spectroscopy measurements and can be applied to studies of the single-molecule thermodynamic properties of DNA origami nanomachines as well as biomolecular complexes. We further demonstrate wavelength selectivity and establish the temperature dependence of the reaction coordinate for base-pair disruption in the shear-rupture geometry, demonstrating the utility and flexibility of this approach for both fundamental studies of local (nanometer-scale) temperature gradients and rapid and multiplexed nanomachine actuation.

Using Optical Tweezers Combined with Total Internal Reflection Microscopy to Study Interactions Between the ER and Golgi in Plant Cells.

Optical tweezers have been used to trap and micro-manipulate several biological specimens ranging from DNA, macromolecules, organelles, to single-celled organisms. Using a combination of the refraction and scattering of laser light from a focused laser beam, refractile objects are physically captured and can be moved within the surrounding media. The technique is routinely used to determine biophysical properties such as the forces exerted by motor proteins. Here, we describe how optical tweezers combined with total internal reflection fluorescence microscopy (TIRF) can be used to assess physical interactions between organelles, more specifically the ER and Golgi bodies in plant cells.

Light-sheet Raman tweezers for whole-cell biochemical analysis of functional red blood cells.

Micro-Raman spectroscopy has emerged as one of the foremost techniques for analyzing biological cells in recent years due to its non-destructive nature and high spatial resolution. The development of optical tweezers has eased the research on biological cells as they confine living cells and organisms in the optical trap without causing much damage. Combining optical tweezers with Raman spectroscopy has opened a wide range of applications in the biomedical field as it facilitates biochemical analysis of biological samples by maintaining in-vivo conditions. Herein, we developed a light sheet-based optical tweezer that traps red blood cells (RBCs) at a very low power density spread across the whole cell, otherwise impossible with conventional optical tweezers. Furthermore, it is combined with micro-Raman spectroscopy to perform whole-cell biochemical analysis for the first time. Raman spectra of individual RBCs recorded under the line focal spot excitation are of superior quality and lack spectral signatures of photo-oxidation and heme aggregation, which is common in point focal spot excitations.

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.

Exploring the conformational dynamics of the SARS-CoV-2 SL4 hairpin by combining optical tweezers and base analogues.

The parasitic nature of the SARS-CoV-2 virus demands selective packaging of its RNA genome (gRNA) from the abundance of other nucleic acids present in infected cells. Despite increasing evidence that stem-loop 4 (SL4) of the gRNA 5' UTR is involved in the initiation of this process by binding the nucleocapsid (N) protein, little is known about its conformational dynamics. Here, we unravel the stability, dynamics and (un)folding pathways of SL4 using optical tweezers and a base analogue, tCO, that provides a local and subtle increase in base stacking without perturbing hydrogen bonding. We find that SL4 (un)folds mainly in a single step or through an intermediate, encompassing nucleotides from the central U bulge to the hairpin loop. Due to an upper-stem CU mismatch, SL4 is prone to misfold, the extent of which can be tuned by incorporating tCO at different positions. Our study contributes to a better understanding of SARS-CoV-2 packaging and the design of drugs targeting SL4. We also highlight the generalizability of using base analogues in optical tweezers experiments for probing intramolecular states and conformational transitions of various nucleic acids at the level of single molecules and with base-pair resolution.

Stable SERS Detection of Lactobacillus fermentum Using Optical Tweezers in a Microfluidic Environment.

Rapid identification of fermented lactic acid bacteria has long been a challenge in the brewing industry. This study combined label-free surface-enhanced Raman scattering (SERS) and optical tweezer technology to construct a test platform within a microfluidic environment. Six kinds of lactic acid bacteria common in industry were tested to prove the stability of the SERS spectra. The results demonstrated that the utilization of optical tweezers to securely hold the bacteria significantly enhanced the stability of the SERS spectra. Furthermore, SVM and XGBoost machine learning algorithms were utilized to analyze the obtained Raman spectra for identification, and the identification accuracies exceeded 95% for all tested lactic acid bacteria. The findings of this study highlight the crucial role of optical tweezers in improving the stability of SERS spectra by capturing bacteria in a microfluidic environment, prove that this technology could be used in the rapid identification of lactic acid bacteria, and show great significance in expanding the applicability of the SERS technique for other bacterial testing purposes.

Controlling the counterintuitive optical repulsive thrust of nano dimers with counter propagating type waves and background medium.

This work focuses on the utilization of counter-propagating plane waves for optical manipulation, which provides a unique approach to control the behavior of Rayleigh and Dipolar nanoparticles immersed in a homogeneous or heterogeneous medium. Our study presents an interesting finding of a repulsive force between plasmonic-chiral heterodimers where the particles move away from each other in both near and far field regions. Interestingly, this repulsive thrust supports the wave like nature of light for the case of homogeneous background but particle type nature of light for heterogenous background. At first, we have investigated the theory underlying the optical trapping of the chiral particle and the impact of this phenomenon on the overall repulsive behavior of the heterodimers placed in air (homogeneous) background. After that, our proposed set-up has further been investigated putting in air-water interface (heterogenous background) and by varying light angle only a little bit. Our observation for this interface case is suggesting the transfer of Minkowski momentum of photon to each optically pulled Rayleigh or dipolar particle of the dimer set, which ultimately causes a broad-band giant repulsive thrust of the dimers. However, in absence of the other particle in the cluster, a single half-immersed particle does not experience the pulling force for the broad-band spectrum. The 'common' reason of the observed repulsive thrust of the dimers for both the aforementioned cases has been attributed to "modified" longitudinal Optical Binding Force (OBF). Technically, this work may open a new way to control the repulsion and attraction between the nanoparticles both in near and far field regions by utilizing the background and the counter-propagating waves. We also believe that this work manifests a possible simple set-up, which will support to observe a background dependent wave 'or' particle nature of light experimentally.

Boosting the Optical Trapping of a Single Virus by Quantum Dots Tagging Increases Virus Polarizability and Trap Stiffness.

Optical tweezers use the momentum of photons to capture and manipulate particles in a noncontact way. Although related techniques have been widely used in biology and materials, research on viruses is still relatively limited. It is hard to optically trap viruses because trap stiffness is rather low and the size of viruses is too small. Here, we used an optical tweezers system coupled with a laser confocal fluorescence imaging system, which allows individual viruses to be imaged and trapped in real time and analyzed using multiple parameters in the culture medium. We show that a single virus tagged by quantum dots (QDs) can increase the real part of polarizability, further increasing gradient force and trap stiffness. With this method, we not only can trap and manipulate viruses in real time but also can analyze their interactions with other targets.

Design of a multi-band Raman tweezers objective for in situ studies of deep-sea microorganisms.

The investigation of deep-sea microorganisms holds immense significance and value in advancing the fields of life sciences, biotechnology, and environmental conservation. However, the current lack of specialized underwater objectives specifically designed for in situ studies of deep-sea microorganisms hampers progress in this area. To address this limitation, we present the design of a multi-band Raman tweezer objective tailored for deep-sea environments. The objective is integrated into a high-pressure chamber capable of withstanding depths up to 1.5 km, enabling in situ microscopic imaging, optical tweezer capture, and Raman detection of deep-sea microorganisms. Through meticulous structural optimization, meticulous material selection, and thorough mechanical analysis of the underwater optical window, the objective exhibits remarkable attributes such as multi-band functionality, extended working distance, and high numerical aperture. Our design yields image quality near the diffraction limit, successfully achieving flat-field and apochromatic performance in each respective wavelength bands. Moreover, the tolerance analysis demonstrates that the full-field root mean square (RMS) wave aberration approaches λ/14, effectively meeting the demands of manufacturing and practical applications. This objective lens constitutes a vital tool for the in situ exploration of deep-sea microorganisms.