Science, Irving K. Barber Faculty of (Okanagan)
Chemistry, Department of (Okanagan)
DSpace
UBCO
An, Minh (Scott)
Master of Science - MSc
University of British Columbia
Optical tweezer is a well-established and continually evolved microscopic trapping and manipulation technique widely employed across numerous disciplines. A tightly focused Gaussian-profile laser can be used to confine homogenous particles by exerting two forces: 1. Scattering force, which maintains the target particles along the axis of the laser, and 2. Gradient forces prevent the particles from laterally drifting off the beam. We constructed a dual-beam variant of the optical tweezer for single-molecule studies. This system allows the simultaneous trapping of two particles, where one serves as an anchor point and the other moves to stretch DNA-based nanostructures. The decoupling from surface anchoring enables the system to achieve sub-piconewton resolution measurement with minimal environmental noise. We developed in-house programs facilitating data acquisition and control of various components. This allows rapid testing and deployment of custom features tailored for different experiment types and DNA structures. Finally, we established comprehensive user protocols and calibrated the system force measurement. The development of this instrument provides a powerful approach for future investigators in single molecular and cellular studies.
CONSTRUCTION OF DUAL-BEAM OPTICAL TWEEZER FOR SINGLE-MOLECULE STUDY by Minh (Scott) An B. Sc. Hons. Biochemistry, The University of British Columbia Okanagan, 2019 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE COLLEGE OF GRADUATE STUDIES (Biochemistry & Molecular Biology) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) October 2024 © Minh (Scott) An, 2024 ii The following individuals certify that they have read, and recommend to the College of Graduate Studies for acceptance, a thesis/dissertation entitled: CONSTRUCTION OF DUAL-BEAM OPTICAL TWEEZER FOR SINGLE-MOLECULE STUDY submitted by Minh (Scott) An in partial fulfillment of the requirements of the degree of Master of Science in Biochemistry & Molecular Biology . Dr. Isaac T.S. Li, Department of Chemistry, Irving K. Barber Faculty of Science Supervisor Dr. Robert Godin, Department of Chemistry, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. David Jack, Department of Chemistry, Irving K. Barber Faculty of Science Supervisory Committee Member Dr. Andrew Jirasek, Department of Computer Science, Mathematics, Physics and Statistics, Irving K. Barber Faculty of Science University Examiner iii ABSTRACT Optical tweezer is a well-established and continually evolved microscopic trapping and manipulation technique widely employed across numerous disciplines. A tightly focused Gaussian-profile laser can be used to confine homogenous particles by exerting two forces: 1. Scattering force, which maintains the target particles along the axis of the laser, and 2. Gradient forces prevent the particles from laterally drifting off the beam. We constructed a dual-beam variant of the optical tweezer for single-molecule studies. This system allows the simultaneous trapping of two particles, where one serves as an anchor point and the other moves to stretch DNA-based nanostructures. The decoupling from surface anchoring enables the system to achieve sub-piconewton resolution measurement with minimal environmental noise. We developed in-house programs facilitating data acquisition and control of various components. This allows rapid testing and deployment of custom features tailored for different experiment types and DNA structures. Finally, we established comprehensive user protocols and calibrated the system force measurement. The development of this instrument provides a powerful approach for future investigators in single molecular and cellular studies. iv LAY SUMMARY An optical tweezer is a tool that utilizes light particles to manipulate tiny objects. Although light particles lack mass, they possess momentum that changes as they travel through different materials with varying refractive indices. This change in momentum generates a force that influences objects upon contact. Despite its seemingly small strength, this force can potentially be used to manipulate small particles, enabling an alternative approach to exploring biological molecules at the individual level. We developed an optical tweezer with a high-powered laser to trap microscopic particles. These particles are coated with various building blocks of life, like DNA and proteins. A motorized mirror is then used to manipulate the particle linearly. By creating a tether between the particle and the molecule of interest, we can stretch/relax the molecule by modulating the mirror. Through this method, we unveil their fundamental physical properties and pave the way for designing and experimenting with novel DNA nanostructures. These nanostructures hold promise for diverse applications in biological research and biomedicine. v PREFACE Minh (Scott) An and Dr. Isaac T.S. Li designed and executed the research work presented. The development team involved three people: Victor Sun with technical consultant, Dyuti Raghu with calibration and Minh (Scott) An with instrument construction. The layout and planning were done with significant help from Dr. Isaac T.S. Li. This work is based on the previously constructed optical tweezer from the Chemla (University of Illinois) and the Bustamente Lab. Victor Sun laid down the framework for the stage control program. Minh (Scott) An developed a data acquisition program and subsequent analysis package. Data analysis was done under Dr. Isaac T.S Li's direction. This work does not include the use of generative AI (genAI). All research data presented is generated by the author and his colleagues, any external materials will be properly cited and referenced. vi TABLE OF CONTENTS ABSTRACT ...................................................................................................................... iii LAY SUMMARY ............................................................................................................. iv PREFACE .......................................................................................................................... v TABLE OF CONTENTS ................................................................................................ vi LIST OF FIGURES ......................................................................................................... ix LIST OF SYMBOLS AND ABBREVIATIONS ........................................................... xi ACKNOWLEDGEMENT ............................................................................................. xiii 1 INTRODUCTION................................................................................................... 1 1.1 Brief overview ...................................................................................................... 1 1.1.1 Raleigh regime .............................................................................................. 4 1.1.2 Intermediate regime ...................................................................................... 6 1.1.3 Ray optic regime ........................................................................................... 6 1.2 Optical tweezer implementation in academic research ......................................... 9 1.2.1 A brief roadmap .......................................................................................... 10 1.2.2 Single-molecule application and beyond .................................................... 12 1.2.3 Optical tweezer applications and considerations ........................................ 15 1.2.4 Instrumentation target and thesis objectives ............................................... 18 2 CONSTRUCTION OF DUAL-BEAM OPTICAL TWEEZER ....................... 20 2.1 Stage 1 - Power control ....................................................................................... 22 2.2 Stage 2 - Beam-splitting and steering ................................................................. 24 2.3 Stage 3 - Trapping and force detection ............................................................... 27 2.3.1 Trapping and stage control.................................................................................. 29 2.3.2 Force Detection ................................................................................................... 31 vii 2.3.3 Live imaging ....................................................................................................... 35 2.4 Instrument enclosure and hardware safety .......................................................... 36 3 DEVELOPMENT OF CENTRALIZED DIGITAL CONTROL ..................... 40 3.1 Data acquisition and steering control program (DAQ-SG) ................................ 41 3.1.1 High-speed, high-bandwidth data acquisition panel ................................... 42 3.1.2 Piezo control allows precise steering trap movement ................................. 44 3.1.3 Integrated shutter control ............................................................................ 46 3.2 Motorized stage control (MSC) .......................................................................... 46 3.3 Particles tracking with CMOS camera ................................................................ 49 4 INSTRUMENT CHARACTERIZATION ......................................................... 51 4.1 Assessment of systemic noises ........................................................................... 51 4.1.1 Laser noise .................................................................................................. 51 4.1.2 Electrical and mechanical ........................................................................... 54 4.1.3 Steering mirror linearity and conjugation ................................................... 55 4.2 Instrument components calibration ..................................................................... 57 4.2.1 Trap stiffness ðð - Power Spectral Density .................................................. 58 4.2.2 Optical sensitivity ðð â Flow calibration ..................................................... 61 4.2.3 Pixel size ð¶ð¶ â Camera calibration ............................................................... 62 4.2.4 Steering sensitivity ðð â Steering mirror calibration .................................... 63 5 PROTOCOL FOR SINGLE-MOLECULE EXPERIMENT ........................... 66 5.1 Multi-flow chamber fabrication .......................................................................... 66 5.1.1 Coverglass preparation................................................................................ 67 5.1.2 Precision-cut channel layout ....................................................................... 68 5.1.3 Fabrication protocol .................................................................................... 70 5.2 Nanoparticles preparation ................................................................................... 72 viii 5.2.1 Nanoparticle washing and handling protocol ............................................. 73 5.2.2 Nanoparticles functionalization protocol .................................................... 75 5.3 Flow circuit assembly protocol ........................................................................... 76 6 SINGLE-MOLECULE STRETCHING OF DNA ............................................. 79 6.1 DNA force-extension measurement protocol ..................................................... 79 6.2 Elasticity of double-stranded DNA ..................................................................... 80 7 DISCUSSION ........................................................................................................ 87 8 CONCLUSION ..................................................................................................... 90 REFERENCES ................................................................................................................ 91 APPENDICES ............................................................................................................... 106 A. Instrument component list................................................................................. 106 B. LabVIEW general schematic ............................................................................ 108 C. Troubleshooting table ....................................................................................... 110 ix LIST OF FIGURES Figure 1. Classification of trapping regime based on object size .......................................3 Figure 2. Basic principle of optical trap in geometrical ray optic regime .........................8 Figure 3. Categories of different optical trapping geometries .........................................11 Figure 4. Schematic of dual-trap optical tweezer .............................................................21 Figure 5. Fully constructed dual-trap optical tweezer with optical path illustration ........22 Figure 6. Schematic and image of Stage 1 - Power control ..............................................23 Figure 7. Mechanism of laser power controls via manipulation of polarized light ..........24 Figure 8. Schematic and image of stage 2 - Beam splitting and steering .........................26 Figure 9. Illustration of beam-splitting mechanism based on polarization angles ...........27 Figure 10. Schematics and images of stage 3 - Trapping and force detection ..................28 Figure 11. Alignment strategy for objective stage ............................................................30 Figure 12. Alignment of motorized stage and microfluidic assembly ..............................31 Figure 13. Illustration of conjugated plane in back-focal-plane interferometry ...............32 Figure 14. Alignment of QPD sensor at the conjugated plane position ............................35 Figure 15. Collimation strategy post-trapping ..................................................................33 Figure 16. Schematic of sample visualization light path and image optimization ...........36 Figure 17. Temperature fluctuations in the instrument room ...........................................37 Figure 18. Environmental noise reduction strategy and hardware safety implementation 39 Figure 19. Electrical components interfacing with central computer ...............................40 Figure 20. Programming flow chart showing input and output signals from computer ...41 Figure 21. Data-streaming function of DAQ-SG program ...............................................43 Figure 22. Steering signal generation function within DAQ-SG program .......................45 Figure 23. Shutters control mechanism and control scheme ............................................46 Figure 24. Technical diagrams for Main Stage Control (MSC) program .........................47 Figure 25. User interface of CMOS sensor program ........................................................50 Figure 26. Laser power measurement during the first hour of operation .........................52 Figure 27. Laser pointing stability assessment .................................................................53 Figure 28. Electrical noise measurement for different state of the instrument .................54 Figure 29. Linearity and timing assessment of the piezo steering motor .........................56 Figure 30. Power spectral density plot of optically trapped particle ................................59 x Figure 31. Trap characterisation .......................................................................................60 Figure 32. Determination of optical sensitivity ðð by flow series assay ...........................62 Figure 33. Schematic for pixel size ðªðª determination method ...........................................63 Figure 34. Schematic for determination of steering sensitivity S .....................................64 Figure 35. Flow cell anatomy and operation ....................................................................70 Figure 36. Visual comparison between different parafilm sealing techniques .................72 Figure 37. Before and after images of particle stock after de-aggregation protocol ........74 Figure 38. Microfluidic assembly .....................................................................................78 Figure 39. Data processing pipeline...................................................................................81 Figure 40. Typical data preprocessing display .................................................................82 Figure 41. Force extension curve series ............................................................................83 Figure 42. Rupture detection and WLC fit .......................................................................84 Figure 43. Histogram plot of fitted dsDNA contour length ..............................................85 xi LIST OF SYMBOLS AND ABBREVIATIONS AD Anti-Dig AFM Atomic Force Microscopy AFS Acoustic Force Spectroscopy ALS Amyotrophic Lateral Sclerosis AOD Acousto-Optic Deflector AOT Angular Optical Tweezer BFP Back Focal Plane BFPI Back-Focal-Plane interferometry BSA Bovine Serum Albumin Bt Biotin CMOS Complementary Metal Oxide Semiconductor CNC Computer Numerical Control DAQ Data Acquisition DAQ-SG Data Acquisition â Signal Generation (program) DDA Discrete Dipole Approximation (theory) FOV Field Of View FDTD Finite-Difference Time-Domain (theory) GLMT Genralized Lorenz Mie Theory HVAC Heating, Ventilation and Air Conditioning (system) kS kiloSample LED Light Emitting Diode MSC Main Stage Control (program) xii MT Magnetic Tweezer ND Neutral Density (filter) Nd:YAG neodymium-doped yttrium aluminum garnet (crystal) NMR Nuclear Magnetic Resonance QPD Quadrant Photodiode PATRRs palindromic AT-rich repeats PCA protocatechuic acid (O2 scavenging system) PCD protocatechuate-3,4-dioxygenase (O2 scavenging system) PDL1 programmed cell death-ligand-1 PD-1 programmed cell death protein 1 PDMS Polydimethylsiloxane PSD Positional Sensing Detector SA Streptavidin shRNA Small hairpin RNA ssDNA Single-stranded DNA SLM Spatial Light Modulator smTIRF single-molecule Total Internal Reflection Fluorescent (microscopy) TBE Tris/Boric/EDTA buffer TDMS Technical Data Management Streaming (file format) TE Tris/EDTA buffer TTL Transistor-Transistor Logic T50M5 Tris/NaCl/MgCl2 buffer xiii ACKNOWLEDGEMENT I would like to thank Dr. Isaac T.S. Li for providing me with an incredible opportunity to work on this project and his guidance on everything academic-related. His insight into electronics, optics, and instrument operations is what made this successful development possible. I thank Victor Sun and Dyuti Raghu for assisting with software and DNA protocol development. Especially Dyuti for her expertise in DNA hairpins. I appreciate that David Bakker and Micah Seung-Hae Yang provided detailed input on controller programs and noise management. Additionally, without their valuable input in the data analysis, it would be impossible to produce results that are faithfully well-matched with the literature. This work is funded by Natural Sciences and Engineering Research Council of Canada (NSERC) and Canada Foundation for Innovation. 1 1 INTRODUCTION The last three decades have witnessed tremendous advancements in the methodology used for single-molecule study. The introduction of novel techniques such as magnetic tweezers (MT), and atomic force microscopy (AFM) has revolutionized single-molecule biophysics research, sparking great interest in the under-explored physical aspect of biological molecules. Combined with ensemble observations, they give researchers a more complete picture of complex systems and phenomena. Another significant milestone occurred in 1970 when Arthur Ashkin created the optical tweezer, a method of manipulating microscale objects with light.1 Since then, the optical tweezer has become an indispensable addition to the single-molecule toolbox and has demonstrated its versatility in a wide range of applications, such as drug development, biomedical analysis and fundamental physics research. This thesis aims to introduce the principles of optical trapping, explain how light can be harnessed to manipulate matter and provide a comprehensive overview of the design, construction, and optimization of an optical tweezer. 1.1 Brief overview The concept of radiation pressure was realized in 1619 by Johannes Kepler during his observation of comet tails.2 As the vaporization of surface materials forms the coma, the gas and dust clouds are pushed away by the pressure from a combination of sun rays and solar winds, allowing them to overcome the cometâs gravity. Kepler noted that the coma always points away from the sun, indicating some underlying mechanical pressure exerted by the sun. This observation is later supported by James Clerk Maxwellâs electromagnetism theory, which predicts the existence of electromagnetic radiation capable of exerting forces along its direction of motion. Since light is a propagating wave of electric and magnetic fields, it can indeed mechanically affect matter.3 âIn a medium in which the (electromagnetic) waves are propagated there is a pressure in the direction normal to the wave, and numerically equal to the energy contained in unit volume.â â Maxwell (1873) 2 In 1901, Nichols and Hull first observed radiation pressure experimentally, as they succeeded in manipulating gas particles using a light source.4 However, it was deemed impractical since the force exerted is insignificant. It was only in 1970, a pivotal year in the history of biophysics, that the development of the laser by Arthur Ashkin demonstrated a groundbreaking application of light. By focusing a laser into a small highly irradiated volume (i.e., the focal spot), Ashkin showed that light can indeed be used to trap microscopic particles. This discovery, that force can be measured by the deflection of light through the trapped particle, opened a new era of single-molecule biophysics with the birth of the optical tweezer. Light, although massless, has energy and is thus able to carry momentum: ðð = âðð(1) where ðð is momentum, â is Plankâs constant, and ðð is wavelength. As photons interact with an object, their momentum changes as the object reflects, refracts, or absorbs light. Due to the law of conservation of momentum, the object must undergo equal and opposite momentum changes in the form of exerted force according to Newtonâs Second Law of Motion. As demonstrated in Figure 1, optically trapping matter can be categorized into three size-based approximations: â¢ Rayleigh regime (âª ðð): trapped object is smaller than laser wavelength, typically ~0.1ðð. â¢ Intermediate regime (â ðð): trapped object size is similar to laser wavelength ~ðð. â¢ Ray optics regime (â« ðð): trapped object is larger than laser wavelength, generally ~10 ðð. 3 Figure 1. Classification of trapping regime based on object size and examples of typical trapping targets. (a) Illustration of a classical optical tweezer: unlike surrounding particles that freely diffuse (orange arrows), a trapped particle can only sub-diffuse within the trapping volume (dashed orange circle) at the beam waist. Radial (blue arrows) and axial (green and magenta arrows) forces prevent particles from escaping. Figure not drawn to scale. (b) The trapping targets are not limited to polymeric particles. Target ranging from atoms in the Raleigh regime to cells in the optics regime can be trapped with different tweezer configurations. The Raleigh and ray optics regimes provide a simplified approximation of the optical force with a clear definition of trapped objects and light components. These regimes are significant in the context of optical tweezers as they form the theoretical basis for understanding and predicting the behaviour of trapped particles. On the other hand, the intermediate regime requires much more rigorous and complex treatment involving fundamental electromagnetic theory, such as Generalized Lorenz-Mie Theory and T-matrix calculations. These methods are required to qualitatively assess the optical force for a wide range of sizes and non-spherical shapes. Direct optical trapping and manipulation of DNA is impossible because of its small size and the native non-spherical conformation (excluding globular DNA). Therefore, functionalized microspheres (0.2-5 µm) are often used as interfaces to which the target DNA 4 is tethered. Typical lasers with a 700-1100 nm wavelength are employed to avoid damaging biological samples. The trapped particles, hence, fall into the Intermediate regime. In literature, ray optics theory is the preferred approach for describing optical trapping theoretically. This is because of its intuitive nature and ability to be visualized via diagrams. To introduce the basic principle of optical trapping, this chapter will briefly summarize the Raleigh and Intermediate regime while elaborating more on the ray optics theory. Understanding the implications of these regimes on the design and operation of optical tweezers is crucial for researchers and academics in the field of biophysics and related scientific disciplines, as it helps in making informed decisions and optimizing the performance of optical tweezers. 1.1.1 Raleigh regime Under the Raleigh regime, a trapped particle is treated as an infinitesimal dielectric dipole with an internal homogenous electric field since its dimension is neglectable. The laser focus is a diffraction-limited region whose diameter is slightly larger than its wavelength. Within this region, light can be described as a gradient electromagnetic field, and the trapping force is thereby proportional to the intensity gradient of this field. The dipole in this regime is assumed to be spherically symmetrical. For successful particle confinement, a tightly focused Gaussian laser beam is required to produce a three-dimensional net force toward the centre of the focusing region. The decomposition of this force yields two components: the scattering force and the gradient force. A stable confinement of dipole within the fields will be achieved when the ratio of the gradient force to the scattering force is greater than unity. The Scattering force is the laser beam's radiation pressure acting on the dipole. As momentum is transferred from the field to the dipole, the radiation force arises as the momentum is conserved during the subsequent scattering process. This change of momentum accounts for the difference between the incoming photon and the re-emitting photon. It quantifies the scattering force as such: ð¹ð¹ð ð ð ð ð ð ð ð = 8ðððððððððððð4ðð63ðð ï¿½ ðð2 â 1ðð2 + 122ï¿½ ð¼ð¼ (2) 5 where ðððððððð is the refractive index of the medium, ðð is the wavenumber where ðð = 2ðððð , ðð is the radius of the particle, ðð is the speed of light, ðð is the effective index of refraction (ððððð ð ððð ð ððð ð ðððð ððððððððâ ) and lastly, ð¼ð¼ is the intensity of the beam. The resulting net force points in the direction of the Poynting vector. It is also worth noting that the material of the particle plays a role in scattering force expression: for a material that has high absorption properties, the arising force is proportional to ðð3 instead of ðð6 as seen in Equation (2).5 The gradient force (ð¹ð¹ððððð ð ðð) is induced by the non-homogenous electromagnetic field of the Gaussian beam. An off-centred dipole will experience a force toward the region with maximum intensity in this field. It can be expressed as: ð¹ð¹ððððð ð ðð = 2ðððð3ðððððððððð ï¿½ðð2 â 1ðð2 + 2ï¿½ð»ð»ð¼ð¼ (3) where ð»ð»ð¼ð¼ is the vectorial difference of the trapping light intensity. Like scattering force and with the assumption that the particle is an extremely small dipole, the gradient force is independent of the particle shape and proportional to the particle radius and, by extension, the volume of the particles: ð¹ð¹ððððð ð ðð,ð ð ð ð ð ð ð ð â 43 ðððð3 . Still, the gradient force is positively correlated to the transverse intensity gradient: ð¹ð¹ððððð ð ðð â ð»ð»ð¼ð¼, instead of the spatially immediate intensity like the scattering force: ð¹ð¹ð ð ð ð ð ð ð ð â ð¼ð¼. Previously, the gradien99t force exhibited in collimated Gaussian beam could only push towards the beam axis (lateral component). Therefore, another counter-propagating beam shooting in the opposite direction is needed to balance the scattering force, i.e., the force in the direction of light propagation. This setup is advantageous in an application demanding high trapping power. The trade-off is the complexity of the setup, which requires great maintenance and calibration effort. Specifically, the calibration of individual lasers and the centring of 2 opposing beams must be carried out for each experiment. A more popular method is utilizing the tight laser focus to produce a gradient of intensity formed along the beam axis, where the laser focus has the highest concentrated optical power. The newly formed axial gradient component counteracts the effect of scattering force. This results in a net force towards the centre of the focal spot in both axial and lateral directions, allowing the particle to be trapped three-dimensionally. 6 1.1.2 Intermediate regime Between the 2 extreme regimes exists the intermediate regime, where neither the dipole nor the ray optics approximations are valid. This regime requires rigorous modelling for the interaction between light and particle for an exact quantitative force description. One must consider the vectorial characteristics of trapping laserâs electromagnetic and partial fields (incident, scatter and internal) and utilize Maxwellâs equation. Considering the trapping of a homogenous spherical particle using a Gaussian beam, trapping force can be described using generalized Lorenz-Mie theory (GLMT), an extension of the classical Lorenz-Mie theory to accommodate for optical tweezerâs tightly focused Gaussian beam.6,7 This theory provides a mathematical representation of the incident and scattered electromagnetic field, thus enabling the calculation of scattering coefficients, which quantify the interactions of the field with trapped particles. This theory also employs several numerical methods, such as Discrete Dipole Approximation (DDA) and Finite-Difference Time-Domain (FDTD), to address different particle shapes and beam geometries. The use of GLMT to calculate optical forces in optical tweezers can be found in great detail through the works of Mazolii8 and Nieminen9. Another successful quantitative approach is calculating the transition matrix (T-matrix), defined by the expansion amplitude of incident, scattered, and internal partial fields.10â13 It allows the calculation of scattering properties such as phase functions, scattering cross-sections and especially scattering coefficients. Similar to GLMT, it utilizes vector spherical wave functions to express electromagnetic fields in a series of multi-pole expansions. Thus, T-matrix offers several advantages: (1) it excels in modelling particles and biological cells with complex geometries, and (2) it can be extended to deal with multi-scattering from particle clusters. Despite their less intuitive nature, these approaches exhibit remarkable versatility in accommodating a wide range of trapped object dimensions and complex trapping scenarios. As a result, they represent a more advantageous approach for quantitatively describing optical force. However, approximative descriptions provide a more accessible understanding and still produce comparable values.14 1.1.3 Ray optic regime Geometric optics offers a viable approach to quantitatively and qualitatively describe the physics governing trapped objects surpassing the wavelength of light in size. Even so, it is 7 essential to acknowledge that several assumptions are made regarding the objects: trapped objects must not only be homogenous and spherical but also non-dispersive and possess a refractive index higher than the surrounding medium. In addition, a more modest electromagnetic field treatment is employed compared to the ones used in the intermediate regime. Light field can be broken down into a bundle of rays while the effect of diffractions and interferences are neglected. This makes it challenging to obtain an accurate quantitative assessment of force for any object less than 20 times the trapâs wavelength.5 Nevertheless, this regime is still commonly used to provide an intuitive interpretation of the basic principle of optical trap. To get a clear picture of optical force in this regime, we consider a minimalistic case of trapping a spherical object fitting all the mentioned physical constraints. A collection of rays representing a Gaussian beam carries a portion of incident beam power. The beam focus can be assumed to be a tight spot. When a ray interacts with particles, it undergoes absorption, reflection (as described by the Fresnel equation) and refraction (governed by Snell's law). The resulting light momentum is changed, and according to the principle of conservation of momentum, an identical change of momentum must be acted upon the object in the opposite direction. The force resulting from the change of momentum can be defined using Newtonâs second law: ?â?ð¹ = ðð?â?ðððð ð (4) where ?â?ð is the momentum of the object, and ð¡ð¡ is time. This force can be broken down into 2 distinct components: scattering force and gradient force (Figure 2). 8 Figure 2. The basic principle of optical trap in geometrical ray optics regime. (a) Scattering force fixes the particle axially at the laser focus. This requires a balance of the forces resulting from the change of momentum of reflected and refracted rays. The incident ray contacts the particle at the angle ð½ð½ and being refracted off at the angle ðð (b) Gradient force confines the particle laterally to the focal spot. Upon particle displacement, a restoring force acts on the particle, returning it to the centre of the Gaussian beam, i.e. the highest intensity region. Lateral force â or gradient force â maintains the particle's position in the X and Y axes perpendicular to the beam axis (Figure 2a). This force is formed by the intensity gradient of a Gaussian laser beam where the intensity is maximal at the beam axis and declines towards the periphery. As the trapped particle drifts away from the centre position, it will always experience a restoring force towards the region of highest intensity, where its potential energy is minimized. The gradient force was expressed by Ashkin15 : ð¹ð¹ððððð ð ðð = ðððððððððððð ï¿½1 + ð ð sin 2ðð â ðð2 sin(2ðð â 2ðð) +ð ð sin 2ðð1 + ð ð 2 + 2ð ð sin 2ðð ï¿½ (5) where ðð is the power of the beam, ð ð and ðð are the Fresnel reflection and transmission coefficient respectively. These factors quantitatively determine the fraction of light being reflected or transmitted for individual ray interaction. Lastly, the angular factor ðð represents the angle of the incident ray, and ðð is the angle of the refractive ray. 9 Axial force â also known as scattering force â keeps the particle in a fixed position along the beam axis (Figure 2b). As the rays interact with the particles, the momentum changes as they are scattered (reflected and refracted) or absorbed by the particle. The incident light gets reflected off the particle surface and loses the forward momentum. This creates the scattering force that pushes the particle forward. On the other hand, light gains forward momentum as it gets refracted upon exiting the particle boundary. The forward momentum resulted in a recoil force in the opposite beam propagation direction. This recoil force is historically insufficient to maintain the position of the particle until a focusing beam is used. Additionally, local heating is expected as the particle absorbs some of the light energy, resulting in a further drop in forward momentum. Therefore, axial symmetry can only exhibit slightly downstream from the beam focus. The scattering force can be written as: ð¹ð¹ð ð ð ð ð ð ð ð = ðððððððððððð ï¿½1 + ð ð cos 2ðð â ðð2 cos(2ðð â 2ðð) + ð ð cos 2ðð1 + ð ð 2 + 2ð ð cos 2ðð ï¿½ (6) Like ð¹ð¹ððððð ð ðð, this force is also proportional to the gradient of light intensity, which in this case, is generated by the laser focus. An ideal three-dimensional particle confinement requires equilibriums of both gradient and scattering forces. Any symmetry breaking due to particle displacement will result in a restoring force towards the higher intensities of the beam. The final net force experienced by the particle is the summation of forces contributed by all the rays that form the beam. Finally, modelling of optical tweezer under the ray optic regime is often restricted to spheroids. Non-spheroid particles introduce additional rotational components (wind-mill effect) and transverse components (optical lift effect) such that ray optics can no longer effectively describe.14 Hence, they require a more complex approach, such as GLMT, as mentioned in section 1.1.2. 1.2 Optical tweezer implementation in academic research In this chapter, our primary objective is to present a concise overview of the wide-ranging applications of optical tweezers in academic research. We aim to highlight its extensive utilization within single-molecule biophysics since its inception in the current cutting-edge 10 applications. Furthermore, we explore the broader scope of optical tweezers by showcasing their versatility and potential across various scientific disciplines. Finally, the landscape of currently available techniques will be discussed, including alternative approaches that address the potential drawbacks of optical tweezers. 1.2.1 A brief roadmap Diverse designs exist for optical tweezers in single-molecule biophysics, ranging from simple single-trap setups to more intricate configurations, including dual-trap or multi-trap arrays and hybrid systems. Traditionally, a biopolymer is linked between a single optical trap and a fixed surface, hence the name âsingle-trapâ (Figure 3a-b). The benefit of this type of setup lies in the simplicity and low setup cost. Thus, it is particularly suitable for experiments emphasizing particle manipulation without requiring high-precision force measurement. The next evolution in trapping geometries stems from the desire to reach the fundamental Brownian noise limit exhibited by the trapped particle. This level of noise isolation proves to be challenging for the single-trap setup due to the mechanical coupling of the target molecule with the interface. Because of this, drift and low-frequency vibrations often mask minute movements of the trapped particles, preventing the system from achieving the highest possible spatial resolution. A dual-trap optical tweezer (Figure 3c) has been designed to address this technical problem.16,17 Since the tethering of the target molecule happens between two optically trapped particles, any drifts experienced are decoupled, increasing instrument stability. Depending on the system of study, the additional complexity and material cost may be well compensated by its superior force measurement capability and stability. Dual-trap optical tweezer has rapidly become the standard design for any research lab in biophysics. 11 Figure 3. Categories of different optical trapping geometries. (a-c) Typical trapping geometries are where the tether is formed between an optically trapped particle and a fixed interface, such as the internals of a flow cell (a) or a particle suctioned by micropipettes (b). The dual-trap design forms the tether between two trapped particles (c). (d-f) Hybrid configurations often consist of optical tweezers combined with Fluorescent microscopy. The fluorescent tag on the tethered molecule is excited and visualized using widefield microscopy (d). For a single trap where the molecule is tethered to the surface, total internal reflection (TIRF) microscopy can be deployed (e). Incorporating confocal microscopy allows simultaneous force detection and Fluorescent measurement (f). Angular optical tweezers introduce additional torque components and directional force (g). With the progress of single-molecule research, there is a growing need for an instrument capable of comprehensively capturing macromolecule dynamics, encompassing three-dimensional changes and interactions among multiple components. This highlights the limitations of conventional setups and motivates developing a new generation of optical tweezers. A hybrid class of optical tweezer instruments, known as "Fleezers," has emerged, integrating various forms of fluorescence microscopy (Figure 3d-f). This integration enables simultaneous mechanical force measurement and fluorescence imaging, offering a powerful toolset for studying complex biological systems at the single-molecule level. Through the observation of epi-fluorescence, thermodynamics of DNA and DNA-proteins interactions were unveiled, showing the similarity of DNA overstretching behaviours to thermal melting18 and 12 revealing the mechanism of Rad51 nucleoprotein - highlighting its crucial role in maintaining genome integrity.19 DNA-proteins interactions study also benefits from hybrid OT-smTIRF (single-molecule Total Internal Reflection Fluorescence) instruments. The behaviour of RNA polymerase under different conditions is explored within DNA curtain assay; optical tweezers allow manual switching of DNA state while smTIRF enables fluorescence measurement of RNA polymerase binding time with minimal background noise.20 On the other hand, OT can measure the unwinding force of DNA translocase along with real-time fluorophore tracking.21 Furthermore, one of the state-of-the-art hybrid setups that are not only capable of spatially resolving in angstrom-scale but also able to detect single fluorophores consists of a combination of time-sharing optical tweezer and confocal microscopy22,23. The system can observe and measure step-wise ssDNA hybridization events, revealing the energy landscape of probe-strand annealing and melting. In summary, the fleezer is a vital addition to the next-generation optical tweezers lineup in pursuit of a complete look into complex biological systems. Lastly, an angular optical tweezer (AOT) utilizes laser polarization to apply angular momentum to a trapped object (Figure 3g). Stable confinement requires a delicate balance of three-dimensional axis and rotational angle. Thus, the most common trapping target is fabricated birefringent cylinders made of quartz where one end can be functionalized for attachment to biopolymers. This allows this variant of optical tweezer to measure not only force and extension but also torque and rotation of the biomolecules. Currently, the single-molecule application centres around DNA-protein interactions16,24 or specific DNA structures like holiday junctions25. Despite its limited scope of application, the angular optical tweezer has been accepted and utilized in diverse research domains, including fundamental physics and nanofabrication, which will be further discussed in the next section. 1.2.2 Single-molecule application and beyond The utilization of optical tweezers in single-molecule biophysics has revolutionized the field by enabling meticulous investigations of individual biomolecules, unravelling their dynamic behaviour, and shedding light on intricate biological processes at the molecular scale. This is because of its exceptional advantages in detection sensitivity and non-invasiveness: it can resolve spatial resolution to sub-nanometres (0.1-2 nm) and temporal resolution in the range 13 of microseconds. These remarkable capabilities have overcome previous technical challenges, facilitating the study of small biomolecules such as DNAs and RNAs. Since then, the physical properties of RNA and DNA have been well explored through classical studies investigating persistence length26,27, major/minor groove binding28, elasticity29â31, etc. Beyond intrinsic properties, secondary structures such as hairpin32, pseudo knots33 and helices34 have also been extensively studied and observed using optical tweezers. The current climate is gearing towards the characterization of DNA-protein complexes such as DNA motor proteins35 and the development of biomolecular machinery like DNA-linked microstructures36,37 and multiplexed DNA origami biosensor.38 For more complex and large biological systems, integrin39 and kinesin40,41 were among the first experiments using this platform, showing the many possibilities in studying macromolecules, especially enzymes and proteins. The recent discovery of force-dependent activity of nsp13 helicase from SARs-Cov-2 playing a critical role in viral replication42 and human hRAD51 nucleoprotein conformational dynamics during homologous recombination event43 further highlights the importance of force in cellular/viral mechanical sensing and response. Thanks to its ability to apply force in a controlled and precise manner, dynamics of protein folding pathway44 and misfolding states45 can be resolved and reveal mechanistic insights into the force-dependent biological process. With the recent COVID-19 pandemic, there has been a growing interest in studying viral proteins, focusing on understanding their intermediate folding states and the mechanisms underlying protein misfolding45,46. This demands continual innovation of this technique, pushing the boundary of spatial-temporal resolution to facilitate the study of these intricate biological systems. Beyond single-molecule biophysics, optical tweezers offer researchers a unique opportunity to delve into a wide range of disciplines, from physics and biology to chemistry and materials science. Its unique versatility stems from the fact that this is a relatively simple and accessible concept, which allows researchers to explore and exploit various aspects within the system, ranging from modification of spatial profile of incident beam to alter light properties. Different beam-shaping strategies allow higher throughput optical tweezers can be achieved that allow simultaneous trapping of multiple particles with holographic optical 14 tweezers which utilize computer-assisted spatial light modulator (SLM)47 or time-sharing module such as galvanometer mirror and acousto-optic deflector (AOD)48. This is particularly useful in the biophysics and biomedical fields as it has been used for automated optical sorting of live cells and polystyrene particles. Features recognition is currently the biggest challenge, which requires integrating other techniques, such as Raman spectroscopy, to sort yeast cells50 or developing a closed-loop automated trapping system based on the diffusion trajectory of target colloidal particles49. Additionally, holographic tweezers have shown great utility in multiplex assays for biomarkers in cancer research.50 An array of confined immune-beads was used to detect the level of carcinogenic antigen and alpha fetal protein corresponding to their luminescent levels. Unlike flow-cytometry platform, holographic tweezers allow long time-scale investigation of a single object while provide a much higher detection sensitivity due to the absence of drastic flow effect. Another well-explored aspect of this technique is changing the polarity of light. Light can be made to carry angular momentum that makes objects rotate around their optical axis in 2 forms: spin angular momentum and orbital angular momentum. This is done using an acousto-optic modulator or Laguerre-Gaussian profile beam.51 Its capability to control and rotate objects also shows great potential in micro-fluidic applications, where mixing is still challenging due to laminar flow at a low Reynold number.52 With recent advancements in microfabrication, an angular optical tweezer can now be used to control microfluidic valves.53 This is done by applying different angular momentum to rotate an optically trapped particle. This particle acts as a hinge of the check-valve structure, and therefore, the structure can be manually re-oriented to block certain flow channels. The incorporation of microfluidic manipulation within a flow cell complements the future advancement of lab-on-a-chip devices. In an attempt to overcome the diffraction limit, an alternative trapping approach has been developed. Plasmonic tweezers show that it is possible to trap nanoparticles using surface plasmons. Plasmon structures are excited by focusing a laser on a metallic surface and generating nanoscale focus near the dielectric-metal interface.54â56 Together with near-field optics, viruses57 and nucleotides58 can be studied, which were previously beyond the capabilities of traditional far-field optical tweezers. 15 1.2.3 Optical tweezer applications and considerations Optical tweezers have become a fundamental tool for investigating the biomechanical properties of secondary DNA and RNA structures, providing detailed insights into their folding, stability, and interactions under various forces and environments. Given their proven utility in probing these complex molecular structures, we have decided to construct a custom optical tweezer system tailored specifically for studying DNA and RNA secondary structures. The secondary structure brings endless biological functions and possibilities which can be observed in nature. It is a well-known factor in neurological disease and cancer, where it plays a critical role in regulating genomic stability and expression59,60. Alternative secondary structures such as hairpins are found to be involved in renal cancer and lymphoma, especially in the repeating segments of gene61. In renal carcinoma, palindromic AT-rich repeats (PATRRs) are non-B DNA structures that commonly exist as symmetrical hairpin structures that vary in length, resulting in varying degrees of propensity. PATRRs mediated chromosomal translocations and were found to be involved with the common fragile site FRA3B - a weak link in the human chromosome where any potential damage from carcinogens and rearrangements can lead to the development of cancer.62 With variants of PATRRs existing in different ethnic groups, their structural stability has yet to be explored. Optical tweezers can be used to gain insight into these hairpins' mechanistic properties, highlighting the importance of factors such as AT and GC contents, length of hairpin stem, and the number and orientation of tandem repeats. In lymphoma, the c-myc regulator gene is constitutionally expressed. This causes over-expression of many genes. Some of these are responsible for the over-proliferation of cells and eventually cause tumorigenesis.63 Specifically, by binding to its promoter, it regulates the expression of programmed cell death-ligand-1 (PD-L1), a checkpoint for anti-tumor immunity. Upregulations of PD-L1 will lead to inhibition of the expression of programmed cell death protein 1 (PD-1) on the tumour cell surface, which allows them to evade T cell suppression. Small hairpin RNA (shRNA) has been shown to be a viable strategy to inhibit the expression of the c-myc gene and hence reduce surface expression of PD-L1.64 For PD-L1 to be a viable therapeutic target using shRNA, hairpin design should have several considerations: sequence specificity, stem and loop length to ensure transcription blockage and binding efficiency, 16 contour length for delivery, etc. Optical tweezers can be employed to optimize the mentioned parameters promptly before moving on to more complex in vitro and in vivo experimentation. In addition to hairpin structure, quadruplex represents another significant secondary structure that is implicated in various cancer and neurological diseases, such as amyotrophic lateral sclerosis (ALS). A common genetic cause of ALS is the expansion of non-coding GGGGCC hexanucleotide repeats in the gene C90RF72.65 These repeats reportedly form stable G-quadruplex secondary structure within the same nucleic molecule (unimolecular) or across multiple molecules (multimolecular).66 The formation of G-quadruplexes has been associated with several genetic challenges, such as disrupting telomere homeostasis by hindering telomerase accessibility, leading to DNA degradation.67,68 Additionally, the bulky conformation of G-quadruplexes can stall replication machinery, resulting in genomic instability.69â71 Unfolding of G-quadruplex structure is required for DNA repair mechanisms, typically facilitated by helicase.72,73 G-quadruplexes can adopt various topologies and conformations, which have been extensively studied using X-ray crystallography and nuclear magnetic resonance (NMR) techniques. However, while they provide high-quality static âsnapshotsâ of different topologies and structures, the dynamic processes such as folding/unfolding and re-arrangement pathways cannot be observed74. Furthermore, external factors like temperature, ionic concentrations and molecular crowding, which significantly influence the stability of these structures, are often overlooked in these analyses. In addition, intramolecular interactions between G-quadruplex with its ligands and shRNA are challenging to study using these techniques. Single-molecule approaches, such as optical tweezers, allow for the direct observation of dynamic processes and interactions in vitro, providing real-time insights into G-quadruplex behaviour. Consequently, these techniques have been increasingly employed to gain deeper insights into G-quadruplex dynamics, contributing to developing potential therapeutic strategies.75 Pseudoknot structure is another structure that has garnered much interest recently. It is involved in various biological processes ranging from viral76,77 to cellular activities78,79. RNA pseudoknots play viral protein expression, particularly the -1 ribosomal frameshifting process. The responsible mRNA sequence motif contains 3 typical regions: a slippery site where translational shift happens, the linker region and the downstream pseudo-knot obstructing 17 elongating ribosomes while positioning them over the slippery site. As frameshifting occurs, it enables the production of RNA-dependent RNA polymerase, a vital protein for viral replication80,81. As a result, pseudoknots have emerged as promising targets for reducing frameshifting efficiency, thereby disrupting viral replication. A distinctive three-stem structure characterizes viral RNA pseudoknot and can adopt various conformations depending on Mg²âº concentrations and the mechanical force exerted by the ribosome as it approaches the slippery site.82 Single-molecule techniques, such as optical tweezers, have been employed to investigate the behaviour of pseudoknots under tension forces comparable to those of the ribosomes. These studies have revealed both threaded and unthreaded unfolding pathways, providing insights into the sequential formation of stems and the stability of different pseudoknot conformers. Threaded topologies, in particular, are currently being explored as a strategy for -1 PRF inhibition: It can be used as a barrier against ribosome, decreases the conformational freedom of the pseudoknot and, finally, the tight junctions between its stem to prevent binding of small molecules which aids in the frame shifting processes.83â85 While optical tweezers offer remarkable versatility and precision in various applications, it is crucial to acknowledge and thoroughly evaluate the accompanying limitations and drawbacks. This is particularly crucial when considering its implementation over other single-molecule techniques. Firstly, investigating individual molecules of interest while producing quantity and quality-rich information often comes at a cost of low throughput. Data reproducibility requires significant effort and careful consideration. This is because the experimental sample size must be large enough to demonstrate intrinsic stochastic behaviour while addressing internal heterogeneity within its population. Consequently, alternative approaches such as magnetic tweezers and acoustic force spectroscopy have emerged as potential solutions to address these concerns, offering significantly higher throughput than traditional optical tweezers. Magnetic tweezers rely on external magnetic fields and magnetic particles to exert force simultaneously on hundreds of molecules of interest.86 On the other hand, acoustic force spectroscopy applies highly parallel forces on multiple biomolecules via acoustic standing waves generated by tunable piezo elements.87 Additionally, 18 particle tracking is essential in both instruments: it determines the throughput and experimental data quality. There have been many advancements regarding tracking algorithms and processing frameworks88, which further increase the output of these approaches. However, the intrinsic trade-offs for these video-based data acquisitions are the lower temporal and spatial resolution than optical tweezers' direct light intensity measurement via photodiodes. Secondly, although an optical-based technique brings unprecedented advantages, optical tweezers face the challenge of limited sample selectivity. The trapping target needs to have a size that is not significantly bigger than the trapping area to maintain good trapping efficiency. This usually ranges from 100 nm (polymeric particles) to 10 µm (bacteria and large viruses). Going beyond the size limits will require a highly specialized setup to overcome other external factors such as gravity and inertia (size >> limit) and detection masking due to Brownian motion (size << limit). Moreover, when planning experiments, one must also consider the potential risk of optical damage. Laser-induced heating due to sample absorption may pose a real challenge in preventing optical tweezer deployment on sensitive biological systems89â92. An alternative technique that does not suffer from the concerns above is atomic force microscopy, which offers much more flexibility regarding sample size and exclusivity. Samples on atomic force microscopy are deposited on the treated surface and thus can be as large as 100 μm, such as osteoblast93,94 and have little to no restriction on morphology with appropriate cantilever design and the ability to do parallelized interrogation. Whereas optical tweezers can apply up to 100 pN of force - suitable for single-molecule applications - single-cell investigations, as well as large DNA structures, may require the application of force in the regime of hundreds of piconewtons95 in which AFM and MT are capable. However, AFM and MT offer only unidirectional manipulation of samples and require extensive sample preparation. These factors make them less suited for studying dynamic biological processes, which are better observed in their natural state using techniques like optical tweezers, which offer non-invasive and complex three-dimensional manipulation capabilities. 1.2.4 Instrumentation target and thesis objectives The optical tweezer system must possess several capabilities to advance our understanding of these secondary structures and assess novel, artificially designed DNA-based molecules. 19 1. Effective manipulation of DNA molecules necessitates the attachment of their terminal ends to two separate interfaces. We have chosen a dual-trap design, where each trap contains a distinct functionalized surface that serves as the anchor point for the two ends of the DNA molecules. This bead-to-bead configuration allows us to decouple interactions, providing improved drift management and superior control over surface chemistry compared to a surface-to-bead interface. 2. Studying the target DNA molecule requires the application of mechanical forces, such as stretching and relaxing. This is achieved by implementing a piezo motor in the light path of one trap, commonly called the mobile trap, while the other trap remains fixed, serving as an anchor. The piezo motor enables the user to move the mobile trap closer to or further from the anchor trap, applying force to the tethered DNA molecule. 3. The instrument must be capable of performing simultaneous high-resolution spatiotemporal force measurements. This capability allows the system to probe the desired DNA structure in real-time, facilitating the dynamic collection of information as events unfold. To achieve this, we incorporated a pair of photodiodes to gather light information from both traps at the end of the optical train. With these three essential criteria for investigating DNA structures in mind, we aim to develop a custom dual-trap optical tweezer system featuring one steerable beam equipped with force measurement capability. The objectives of this thesis are, therefore, fourfold: (1) to construct the dual-trap optical tweezer instrument, (2) to develop a centralized digital control system, (3) to establish calibration protocols and single-molecule experiment procedures, and (4) to validate the instrument by conducting a classical hairpin pulling study and comparing the results with existing literature. 20 2 CONSTRUCTION OF DUAL-BEAM OPTICAL TWEEZER Optical tweezer systems can be pre-build or home-build. The first approach offers convenience and established technical support as a trade-off for high start-up costs and limited customizability. Most single-molecule research groups prefer the latter option for their specialized applications despite the substantial instrument development and training effort. Although the home-built optical tweezer system is well-documented and has a relatively straightforward optical train, its complexity comes mainly from integrating various electrical components. An excellent approach to remedy this challenge is to build the trap using a commercial microscope platform, typically from experienced manufacturers like Nikon or Olympus. This reduces the overall complexity of the setup at the cost of limiting its capability to simple trapping and measurement. We opted to construct the system from scratch as this allows total control over every aspect of the system, from sample visual and laser detection to environmental isolation and safety implementation. Our design is a modified version mentioned in Bustamanteâs manual.96 The instrument consists of 3 main sub-systems: trapping, visualization, detection and data acquisition. The schematic of the instrument is illustrated in Figure 4, and a detailed list of components is included in the appendix. 21 Figure 4. Schematic of dual-trap optical tweezer. The instrument comprises 3 stages: (1) a 1064 nm collimated laser is passed through an isolator to prevent back reflection and followed by a half-waveplates contraption which allows the user to control total power and power distribution between traps; (2) the beam is split to 2 orthogonally-linearly polarized beam where one beam is steerable; (3) the beams enter the objective, forms two trap at laser focus before being collected by the rear objective and sent to photodiodes at the end of the optical train. Additionally, a separate LED beam is used to visualize the sample stage. The beam is captured downstream using a CMOS sensor. An overall technical goal of the optical tweezer instrument is to have a correctly sculpted trapping beam(s) entering the trap-forming objective lens and deliver the output light to the detectors. Therefore, components within this system are grouped into stages defined by their overarching functionalities. The stages also serve another purpose: they act as breakpoints during construction. Validation of beam profile and beam centration was carried out upon completion of checkpoints to ensure optimal alignment between stages. This chapter will address key functionalities and critical alignment steps for each stage. The figure below (Figure 5) depicts the laser beam path through each stage (red split into purple and yellow, colour-coded based on the polarization state) along with the separate LED path (blue). 22 Figure 5. Fully constructed dual-trap optical tweezer system with optical path illustration. Every component was carefully positioned and aligned based on a reference iris. Thoughtful consideration was given to cable management and space allocation. Although the assembly process typically proceeds from upstream to downstream, there were instances where components from multiple stages were assembled simultaneously due to the shared beam paths. 2.1 Stage 1 - Power control Stage 1 involves the deployment of a high-powered laser and associated beam-shaping components (Figure 6). For the laser system, two pivotal considerations must be accounted for, depending on the study's requirements: the laser mode and the wavelength. Single-molecule investigationâs common choices range from 830 nm to 1064 nm laser97,98, and these have been shown to cause minimal damage to the live sample89,92. A typical optical tweezer setup employs a continuous wave (CW) laser due to its relatively low complexity level and reduced opticution risk. However, there has been a rise in the usage of pulse lasers recently due to better signal-to-noise ratio and better sensitivity.99,100 Considering other factors such as affordability and availability, we used a single-mode ND:YAG laser (VCFL-20-00120, Spectra-Physics) with an operating wavelength of 1064 nm and a Gaussian beam profile (TEM00). The laser module comes with a collimator used as a construction starting point. The optical train height is determined to be 81.3mm from the surface, with the objective stageâs operational range being the limiting factor. Then, a set of irises is calibrated to the same height, serving as an alignment reference for the construction process. 23 Figure 6. Schematic and image of Stage 1 - Power control. Telescope system 1 (T1) decreases the beam diameter to a factor of 0.625 to pass through the Faraday isolator. The initial unpolarized beam is cleaned using a plate polarizer. Subsequently, the polarization angle is manipulated by a half-wave plate contraption to modulate trapping power. This stage also includes a Faraday isolator (ISO-FRDY-08-1064-N, Newport) to prevent back reflection from damaging the laser source. This is being set up behind a telescope system T1 that reduces the beam diameter to 4 mm (demagnification ratio 0.625x) to match the aperture of the isolator. Next, the laser power is further fine-tuned by a system of two half-waveplates flanking a beam splitter. Since a stable laser source is required to obtain highly sensitive optical trapping, one common practice is to use a high-powered laser and discard most of its power via beam dumps. As half-wave plates alter the angle of linearly polarized light, the resulting beam can be broken into two sub-components: s-polarization (90â° or perpendicular to incident plane), p-polarization (0â° or parallel to incident plane). The output polarization angle determines the amplitude of each component. Thus, the first halfwave plate 24 facilitates the power control process, rotating the input polarization such that only a certain amount of 0â° polarized light passes through the middle beam splitter. (Figure 7) Subsequently, the second halfwave plate can be manually tuned for a different purpose â to control the power distribution between the two traps downstream. Figure 7. The mechanism of laser power control is via the manipulation of polarized light. The incoming unpolarized light from the laser collimator can be broken down into S-polarization (90â°, at the right angle with the plane of incidence) and P-polarization (0â°, parallel to the plane of incidence) components. The S-polarization is reflected off the plate polarizer, allowing linear P-polarized light to be transmitted. Next, the Faraday isolator allows one-way transmission of light via the rotation of linear polarized light so that any back-reflected light will no longer have the same polarization and thus can no longer transmit upstream through the filters within the isolator. Finally, A system comprising two half-wave plates and a beam splitter is used for (1) shown as a green double arrow - adjusting total input power for trap formation by rotating P-polarized light (Æðð) to reintroduce a certain amount of S-polarization light and then direct it to the beam dump via the beam splitter; (2) shown as a blue double arrow - equalizing laser power between traps by redistributing S-P polarization to 50-50 using a second half-wave plate (Æðð = ððððâ°). The laser power is monitored using a power meter after the beam exits the last half-wave plate. Power checks for individual traps can be performed further downstream when the beam has been split. Typically, we maintained 1.6W of total power, which was split into 800 mW for each trap. 2.2 Stage 2 - Beam-splitting and steering It is established that single-trap configuration often suffers from low-frequency vibration and mechanical drift. Other types of drift, such as pointing drift from the laser and z-drift (drift along the optical axis as optical components heat up), further complicate calibrations and feedback attempts. On the other hand, by using an additional trap acting as the other anchor, the dual-trap system has the advantage of having the entire interaction of interest decoupled 25 from the surface, thus increasing the overall stability. Additionally, the noise is tied from one trap to another as they pass through the same optical elements, reducing the subsequent calibration protocol's complexity. This stage defines the difference between single-trap and dual-trap configurations. The main beam is split into 2 separate beams and joined back to the same path using a pair beam splitter (PBS10-1064, Thorlabs). Then, they are expanded to the final diameter of 6.4 mm using the T2 telescope system and reflected to stage 3 using a dichroic mirror. This stage also includes the setup of the additional LED light source ((LED660L, Thorlabs) for sample live imaging. The LED beam passes through the same dichroic mirror, heading to stage 3 along with the reflected laser beams. This can be done by marking the rear aperture location on the table and aligning the focal point of the LED beam accordingly. Once the system is fully constructed and operational, the LED mirror and lens should be adjusted using trapped particles as an imaging reference. For optimal particle tracking, the live imaging area should cover as much of the CMOS sensor as possible while ensuring that the particle shape and illumination are of sufficient quality for monitoring purposes. 26 Figure 8. Schematic and image of stage 2 - Beam splitting and steering. The stage contains a beam splitting and a telescope system (M=1.6) to expand the beam to its final size before entering the trap-forming objective in the subsequent stage. An additional LED light path is included to visualize the trapping plane during the experiment. The incoming beam is split into 2 orthogonally polarized (s and p) beams using a cube beamsplitter. The s-polarized beam (90â°) will act as a mobile trap as it is reflected off a piezo steering mirror, while the p-polarized beam (0â°) reflects off a fixed mirror and becomes an anchor trap. (Figure 9) One major advantage of utilizing differently polarized light for the traps is that there will be no interference between the traps. It will allow traps to be monitored individually at the detection stage. The components were arranged closely in a tight cluster to minimize per-beam noises caused by air fluctuations. Another critical consideration when aligning this stage is to ensure the steering mirror was set up at a correct conjugate plane position for back-focal-plane interferometry, which will be discussed in detail in the subsequent stage. 27 Figure 9. Illustration of beam-splitting mechanism based on polarization angles. The incoming linearly polarized beam (red) with a polarization angle Æðð is broken down into S or 90â° and P or 0â° polarized components. The S beam (yellow) is transmitted through the beam splitter and reflected off the fixed mirror, while the beam splitter reflects the P beam (purple) into a mirror mounted on a piezo steering motor before rejoining the same path as the S beam. Because of the split path, the beams can be slightly misaligned such that they will not overlap completely, allowing them to form 2 separate optical traps in stage 3. To help with the analysis process, the nomenclature A and B refer to fixed trap and steering trap, respectively. 2.3 Stage 3 - Trapping and force detection Once the beams are sculpted via the previous two stages, they enter the last stage of the instrument, where experimentation can be carried out. The optical components in this stage are responsible for 3 essential operations: trapping and stage control, force detection and live imaging. (Figure 10) Before construction, careful planning is needed to position other components, such as cables, power supplies, control modules, and microfluidics pumps, to minimize noise while ensuring ample working space for future maintenance/troubleshooting operations. 28 Figure 10. Schematics and images of the fully constructed stage 3: Trapping and force detection. This stage contains the trapping light path and the LED light path. The trapping light path is terminated at the quadrant photodiode sensors, while the LED light path is stopped at the CMOS camera. This stage also includes a variety of line filters and neutral density (ND) filters to ensure optimal performance and protection for the respective sensors. 29 2.3.1 Trapping and stage control The sampling stage consists of 2 identical water-immersed objectives (UPLSAPO60X, Olympus) mounted on separated ultra-precision stages (ULTRAlign, Newport). Using water immersion objectives has several advantages. Firstly, matching refractive indices between the immersion media and sample buffer (aqueous-aqueous) results in less spherical aberrations at the depth of the chamber and higher trapping stability.101 As a result, a consistent optical pressure is observed over a great range of sample depth (~150 μm, compared to ~20 μm from the oil objective). This allows the sample to be trapped away from the surface to avoid undesired hydrodynamic effects. Index matching also prevents focus degradation102 and elongation, often occurring in oil-immersion objectives.103 The alignment of the objective lens is paramount in attaining high-performance optical traps. Considering six degrees of freedom (Figure 11), the objectives must be positioned perpendicular to the optical axis to ensure minimal light loss. This alignment minimizes variations between the two traps and maximizes the optical power delivered to each trap. The objective stages were first positioned with a pinhole mounted on the holder serving as the reference and a calibrated iris downstream. (Figure 11b) The beam should pass through the centre of the calibrated iris. Both stages for front and back objectives should be performed during this time. Once the position of the ULTRAlign stages is fixed, the pinhole can be replaced with the front objective. Since the holder is fixed with a single standard 8-32 setscrew, we notice a significant variation in the pointing angle between each installation attempt. A possible way to ensure the correct placement of the front objective is to place a calibrated iris about 7 mm away from the front lens. Then, the beam should be trimmed to a third of its original diameter, forming a small clear spot on the calibrated iris. Now, the alignment of the front objective can be attempted. (Figure 11c). 30 Figure 11. Alignment strategy for the objective stage. (a) The objective stage assembly consists of the positioning stage and the objective holder, each with multiple degrees of freedom. (b) First, the positioning stage is secured onto the table and aligned by a mounted iris. This allows a rough estimate of the positioning of the stage with regard to the beam path. (c) the objective can now be mounted and aligned. It is found that the alignment of both components requires careful attention to not only the 3 main XYZ axes but also the pitch α and the yaw β rotation. Lastly, the microfluidic sample is mounted on a custom-made U-shaped bracket slotted into a vertical rail so that the chip will slide into the objectives gap of 4mm (Figure 12). The motorized translation stage and 3 servo actuators (Z825B, Thorlabs) provide precise sample manipulation with a resolution of 0.05 µm. During the installation, an important note is to ensure that the microfluidic chip is perpendicular to the beam path to minimize aberration from the tilted stage. This can be fine-tuned later by trapping a microsphere and dragging it laterally on the inner surface of the chip while observing for the focus shift. There should be no change in the bead images if the stage is well-aligned. Furthermore, due to its design, the chip bracket imposes a horizontal spatial constraint of 5 mm. The installation of the motorized stage should be carried out with the pre-assembled microfluidic sample to determine the optimal position that accommodates all requirements and constraints. An eventual stiffness calibration for trapped particles will also reveal any misalignment of the chip and, by proxy, the translation stage. 31 Figure 12. Alignment of motorized stage and microfluidic assembly. The 3-axis motorized stage was positioned so that the chip was perpendicular to the optical path and in-line with the microfluidic channels. A rail system guides the flow cell into the gap between the objectives, with a stopper at the bottom of the rail to prevent collisions with the objectives as the sample arm is lowered. This method enables the placement of the flow cell with minimal positional variation from one experiment to another. 2.3.2 Force Detection Previously, an additional weak laser is employed to monitor the particle position. This approach has some benefits, such as having linear response decoupled from trapping potential and better range.104 However, there is a significant drawback: this configuration requires a separate optical path and thus requires independent calibration for environmental noises. There is an increase in the complexity of the setup, planning, and experiment protocol. Hence, we utilize the back-focal plane interferometry (BFPI) method105,106, which allows the same trapping light to be used for particle measurement. By capturing all the light passing through the trap, we can monitor particle displacement directly via the change in the interference pattern. A projection of the beam image is extended from the steering mirror in the beam-splitting stage onto the final component at the end of the optical train, the position detectors. The projection is delivered by ensuring components along the optical train are aligned to each other's focal planes, thus extending the conjugate planes from the steering mirror along the beam path and eventually to the photodetector. The photodiode detector must measure the light pattern that appeared on the conjugate plane, as this pattern reflects the particle motion within the trapping plane but not the movement of the steering mirror. (Figure 13) 32 Figure 13. Illustration of the conjugated plane in back-focal-plane interferometry. The beam image projected onto the steering mirror follows the optical path to the photodiode sensor. In (a), the components highlighted in bold are conjugated, meaning the images at these positions are mirrored. In (b), the conjugated image remains stationary as the steering mirror moves while the rest of the optical path changes. As a result, the QPD measurement remains unaffected. Therefore, when the beam motion becomes negligible at the conjugated plane, any observed changes in the beam path must arise from particle motion. Extension of the conjugate plane past the back focal plane of the rear objective was done using a pair of relay lenses. First, the output beam after the back objective is collimated. This should be done before every experiment and verified by (1) checking beam sizes along the optical axis using a Near Infrared Sensor card (Newport, F-ICR1) and (2) checking attenuated beam patterns via a CMOS camera, as seen in Figure 15. Once the collimation is carried out, the beams are split into separate paths where the corresponding relay lens will then extend the trap image to the subsequent detectors. 33 Figure 14. Collimation strategy post-trapping. A collimated beam is essential for conjugating the beam image plane (red line) towards the sensor. (a) the 2 ways to check for beam collimation are visual inspection using the detector card and obtaining a focused beam image with the CMOS sensor aligned correctly. (b) Checkpoint 1 shows the near-far alignment of the laser spot. The laser spots on the detector card should be identical in size regardless of the inspection location. (c) Checkpoint 2 utilizes live images of both traps in focus. They should exhibit sharp beam images, indicating that the beam before the focusing lens is well-collimated. 34 Two types of detectors are employed for displacement measurement: high-response photodetectors, quadrant photodiodes (QPD) and position-sensitive detectors (PSD). Our system uses quadrant photodiode detectors for more accurate and sensitive measurements22,23. As the QPD captures the light at the end of the optical train, its four quadrants (A, B, C, D) convert photons to electrical current and perform analog computing within the integrated circuit. The quadrants are grouped into 2 groups, and the difference in the sum voltage of these groups is calculated and sent to the data acquisition card: ððð£ð£ððððð ð ððð ð ð ð ðð = (ððð´ð´ + ðððµðµ) â (ððð¶ð¶ + ððð·ð·) (7) ððâððððððððððððð ð ð ð ðð = (ððð´ð´ + ððð¶ð¶) â (ðððµðµ + ððð·ð·) (8) where ððð£ð£ððððð ð ððð ð ð ð ðð and ððâððððððððððððð ð ð ð ðð is the voltage difference due to the change of interference pattern across the sensor in X and Y direction which represent, by proxy, the raw displacement of the trapped particle. Each QPD is mounted using a custom fabricated bracket mounted on a 3-axis linear translation stage. Aligning QPDs in the X and Y axes with respect to the beams requires both horizontal and vertical voltage differences to be zero. This means that the trapping beam is at the center of the QPD, where its sensitivity is highest and non-biased. Finally, we proceeded to align the QPD to the conjugated plane. While steering the mirror sinusoidally, we slowly move the QPD along the optical axis and observe the signal collected via an oscilloscope. The location at which the sine wave is minimized is the location of the conjugate plane. (Figure 14) This correlation stems from the direct relationship between image relay quality and the detected steering motion via the QPDs. As components approach their corresponding conjugate planes, the impact of steering motion on the detection signal decreases, resulting in improved resolution of particle detection. However, the QPD signal ceases to further decrease at the zero-to-peak value of 20 mV (1.4 nm) at the max steering range. This indicates a misalignment of conjugated plane assembly upstream. Considering the typical steering range of 2 to 4 V (out of 10 V max range) during the experiment, the maximum positional variation is ~0.55 nm. This was deemed acceptable, and the accessibility of conjugated plane assembly allows future optimization should the need arise. 35 Figure 15. Alignment of QPD sensor at the conjugated plane position. A 10Vpp sine waveform is applied to the steering mirror, and its position is recorded (lower plot, orange). The QPD sensor is centred on the optical axis Z and positioned at three locations along this axis: 25mm, 12.5mm, and 0mm away from the conjugated plane (upper plot, blue). As the sensor approaches the conjugated plane, the signal is minimized. A 20mV (1.4 nm) residual movement is observed when the QPD is placed at the determined conjugated plane position. 2.3.3 Live imaging Visualization of the trapping laser and the trapped bead is done using a separate light source, providing Kohler illumination of the trapping region. This is done by focusing a blue LED onto the back focal plane of the front objective (Figure 16a). The resulting collimated light illuminated the entire region of interest within the spacing between the objectives. Upon exiting the back objectives, the output light passes through the dichroic mirror (M254H45, Thorlabs) and heads straight to a CMOS sensor (DCC1545C, Thorlabs) through a series of focusing lenses and low-pass filters. Tuning the image quality is essential for trapping operations, as bad imaging would make tracking and manipulating particles difficult for the user. A sharp and symmetrical bead image is useful in differentiating single beads and bead aggregates during trapping operation. Furthermore, it also negatively impacts the calibration of the camera and steering mirror. To achieve the best image, the particle image should be fine-tuned by adjusting the camera along the optical axis during the experiment to ensure even illumination of the trapped particles (Figure 16b). 36 Figure 16. Schematic of sample visualization light path and image optimization. (a) Blue LED is sculpted to illuminate the sample plane (small illustration). (b) live images of two trapped particles 2.4 Instrument enclosure and hardware safety High-resolution optical tweezers face a set of environmental challenges that should be considered as early as the planning phase of the project. Pointing stability - the ability of the laser to point at a fixed target over time is heavily influenced by how the laser is constructed and the quality of the optical train. There are 3 major factors that need to be addressed: temperature, airflows and mechanical vibration. 37 Temperature change alters light propagation through the assembly since it can lead to deformation and mechanical stress of the optic elements, causing drift and misalignment. The temperature fluctuations could result from changes in ambient temperature and moisture within the instrument space throughout the day. Thus, the instrument was built and operated in a temperature-controlled environment with an HVAC system set to 22 degrees Celsius. Temperature was monitored over one week with a 1-hour interval. The temperature is between 0.5-1 degrees Celsius within a week, with an hourly variation of 0.1-0.2 degrees. (Figure 17) Figure 17. Temperature fluctuations in the instrument room. The temperature is monitored hourly over a week, and the central HVAC system maintains a relatively constant room temperature. However, starting from the 83rd hour, a significant temperature drop occurs, likely attributed to a sudden regional temperature decrease that challenges the HVAC system's ability to compensate. This observation highlights an inherent limitation of centralized conditioning systems. To address this issue, it is recommended that a separate HVAC system or an airtight enclosure be implemented. Airflows can significantly impact the stability of laser pointing, with a more pronounced effect observed when multiple optical components are exposed to the environment. To mitigate this issue, we built custom enclosures using aluminum extrusion and black cardboard to cover most components except the sampling stage. We excluded this stage as its component; the two objectives are placed closely enough that no trapping laser is exposed to the air. Moreover, this also allows easy access to install a microfluidic chamber during experimentation. So far, there 38 are no observable impacts on laser stability from airflow, and full containment of this stage is planned for future upgrades. Similarly, mechanical vibrations contribute to the instrument's baseline noise, effectively reducing its force detection resolution. Building the instrument in underground laboratory space and on an optical table helps filter out or dampen ground vibrations, which usually excite at 1-3Hz. We used air-spring isolators (S2000-A, Newport), which excel at filtering out frequency in the range of 1-30Hz. A basement environment is highly recommended, as it reduces temperature fluctuations and floor vibrations. Unfortunately, our instrument was placed on the top floor (FIPKE building, UBCO). Thus, further characterization of ground noise should be performed. (Figure 18) Another source of vibration comes from the form of acoustical noise. This was combatted with the installation of acoustic foam surrounding the instrument and the custom enclosures mentioned above. Additionally, all electrical cables are wrapped in cotton and clamped down to the table to reduce the effect of air draft. Lastly, there are several other good practices we have implemented to ensure optimal performance and safety of our instrument: (1) Use a fixed-height pedestal made from a low thermal expansion coefficient material such as solid stainless steel to support vital optical elements. A height-adjustable pedestal system is used for elements that require periodic maintenance and alignment. (2) Removing as many unnecessary components (laser module, piezo controller, power supply, data acquisition card, etc.) from the optical table as possible to avoid additional noises. (Figure 18a) (3) Limits human contact with the instrument, especially during experiments, to prevent the introduction of unnecessary air fluctuations and mechanical drift. (4) Custom-built limiting switch that audibly warns user from âcrashingâ the chamber into the objectives (Figure 18b-d) (5) The addition of an uninterruptible power supply allows users to quickly save and turn off instruments in case of power disruption, while a surge protector guards sensitive components from being damaged in the event of a power surge. 39 Figure 18. Environmental noise reduction strategy and hardware safety implementation. (a) The instrument room is covered in noise-isolation foam. Additionally, the instrument was isolated from ground vibration using a combination of mechanical isolators and an optical table. The custom enclosure is fabricated to minimize the effect of air fluctuation further. Most components that were able to produce mechanical noise were removed from the table. (b) Custom-built safety module with end-switch produces audible and visual alerts using a speaker and LED to warn users about potential stage crashes. (c) physical limits set by the microfluidic bracket, the red lines and associated directions show the scenario where the trap-forming objectives may crash if the user exceeds the set limit. (d) The limit switches are installed to warn the user of a potential crash. 40 3 DEVELOPMENT OF CENTRALIZED DIGITAL CONTROL One of the major challenges when it comes to a home-built optical tweezer is to program software that interfaces with all the electrical components within the system. Components come from different manufacturers and are often offered a variety of approaches, ranging from physical interfaces (Piezo controller â NPC3SG, Newport; stage controller â KDC101, Thorlabs) to digital interfaces (CMOS sensor, laser module) in the form of proprietary software or integration library. Custom software development allows total control over our instrument, from additional features to code maintenance, catering to our specific needs. We chose LabVIEW® as our development platform due to the well-supported industry integration and abundant reference materials from its community. Each component is assigned specific roles (Figure 19), and a central computer handles their operations. While components with digital interfaces can be connected directly to the computer, components with analog input/output (I/O) require a high-bandwidth multifunction data acquisition card (USB 6212, National Instrument) to modulate and receive signals. We have developed two LabVIEW® programs for the primary functions of the optical tweezer system, including measurement and control. Figure 19. Electrical components were interfacing with the central computer. While the camera and translation stage allows direct digital connection, others are analog controls requiring analog-to-digital conversion via a data acquisition card. 41 We split the centralized control into separate programs to ensure optimal reading/writing rate when recording data and prevent complete crashes if certain system elements ran into problems. Other parts of the optical tweezer system come with a fully built controller program that is well-designed and packed with a variety of features, namely the laser control (VGEN), camera CMOS control (ThorCam) and microfluidic pump control (Pumpkintrol). To save development time and effort, we opted to use these existing programs as they are. This chapter will provide detailed insights into the Main Stage Control (MSC) and Data Acquisition-Signal Generation (DAQ-SG) program and briefly discuss the camera program. Figure 20 below shows the data flow from different components within the instrument. Figure 20. The programming flow chart shows input and output signals from the central computer. A single central computer is used to control every aspect of the instrument. Analog components are communicated via a data acquisition card. In contrast, components with digital interfaces such as laser module, CMOS camera and motors for the sample stage can be communicated directly to the computer via standard protocols such as USB, RS-232. 3.1 Data acquisition and steering control program (DAQ-SG) Within our system, we have designed a versatile LabVIEW® program that serves as an interface for the data acquisition card. This card efficiently manages the simultaneous collection of voltage data from the photodiode sensor while generating analog signals for the 42 steering mirror. The multifunctionality of this setup simplifies our interface requirements, centralizes data management, and streamlines the process for future development endeavours. The DAQ-SG program boasts two primary functions that are pivotal in our optical tweezer setup. Firstly, it facilitates streaming raw and binned data from the DAQ card to computer storage. This function ensures the seamless acquisition and storage of critical experimental data. Secondly, the program tasks the DAQ card with producing fully user-customizable waveforms for the steering mirror. This allows precise manipulation and control of the optical trapping process. In the upcoming chapter, we will discuss the overall technicalities of these functions. 3.1.1 High-speed, high-bandwidth data acquisition panel Our detection sensors QPD are made of 4 quadrants containing arrays of photodiode elements; the difference signals are voltage analogs of the changes in light intensity from the incoming beam. A high-speed, high-bandwidth data acquisition card (USB-6212) is needed to facilitate an analog-to-digital interface and allow the data to be stored digitally in the central computer. DAQ-SG program was developed to communicate directly with this card. Voltage data were streamed back into the computer at an aggregated rate of 400 kS/s (50 kS/s per channel, 8 channels). As they belong to the same National Instrument ecosystem, LabVIEW® offers a robust suit of functions and documents and a proprietary file streaming format Technical Data Management Streaming (TDMS), allowing high-bandwidth read/write operation. We designed our programs using the Finite State Machine principle. By breaking the operations into four individual stages, we reduce unnecessary computational resources hogging from non-essential functions (refer to Figure 21): (1) IDLE: Load settings and configurations. The user can adjust and modify parameters freely within this state, from file name, and channels to record, acquisition rate etc. (Default state) (2) SETUP: Initiate specified tasks based on input parameters, then move on to the next state. This state only runs once for every userâs specified task. (3) RUN: Perform the tasks until user intervention or timer runs out. (4) STOP: Clear the finished tasks from memory, close the written file, and return to the idle state. 43 Figure 21. Data-streaming function of DAQ-SG program. (a) the states of the function and their respective tasks. (b) Streaming functions start at an idle state when the program is initialized, waiting for the user trigger. The function will spend most of the time in either IDLE or RUN state. During the RUN state, the function can switch on RECORD options upon user request. This allows the data stream (light blue) to be written to physical storage before being discarded. (c) The user interface of the DAQ-SG program, highlighted portion, is the data-streaming function. Data acquisition has 2 modes: raw and average. In raw acquisition, raw data were streamed and recorded into data storage as is. This allows users to record data at a sampling rate between 100-400kHz. This allows the user to resolve discrete steps made by the trapped particle in the scale of microseconds to reach the Brownian motion limit. The resulting files usually contain millions of samples and are hundreds of megabytes, making it a challenge for storage and analysis. Hence, we only use this mode to calibrate a short interval dataset (30-60s) for trap stiffness. The raw data undergoes a binning process for normal force measurement operation before storing it. The user determines the binning factor (or the averaging factor). We found that averaging our sampling frequency down from 50kHz (raw) to 1kHz (averaged), a factor of 50, is satisfactory for standard operation. Lastly, the acquisition panel also features a variety of quality-of-life functions such as auto file indexing, duplicate name check, timer, independent channel settings, etc. 44 3.1.2 Piezo control allows precise steering trap movement The steering piezo mount (PSM2SG, Newport) enables precise mirror movement of up to 0.04µrad, with a built-in closed-loop feedback feature ensuring high repeatability. The accompanying controller module (NPC3SG, Newport) produces a low-noise, high-resolution signal for the steering mount based on analog input from the user. The DAQ-SG program is equipped to stream data and generate drive signals for the steering mirror to consolidate instrument control. The signal generation panel contains three ways for the user to control the steering mirror and, by extension, the mobile trap. Basic mode offers several pre-set signals (sine wave, rectangle, triangle) with adjustable frequency and amplitude. In custom modes, the user can specify the exact signal to be sent to the mirror controller. On the other hand, manual mode allows the user to control the steering mirror in real time fully. To reduce loop execution time, the signal generation function must be built in a separated operating loop. The function contains its own similarly designed state machine with 4 states mentioned in the previous chapter. (Figure 22) 45 Figure 22. Steering signal generation function within DAQ-SG program. (a) all possible states of the function and their main task. (b) loop operation of the function. Upon user trigger, the program will escape the idle state and move into the offset stage, constantly outputting a DC signal at a user-specified voltage. At this state, any additional user trigger, including âstop function,â or âstart steering,â or âchange value,â will initiate a loop reset (grey line). The function will go through the loop (STOP > IDLE > SETUP) and construct a signal based on new parameters before outputting it to the steering controller (blue line). (c) User interface portion of the Signal Generation function (highlighted). Under normal operation, the traps are levelled (i.e. positioned at the same height). Due to small thermal drift, it was observed that the trap drifted by up to 1 micron from day to day. Hence, users may have to adjust the alignment to ensure they are correctly positioned slightly. We implemented compensation/offset settings where the user can reposition the trap without manually adjusting the optical components. (Figure 22b, OFFSET stage) This also comes with a small sacrifice of a few microns in both X and Y steering ranges, as they are reserved for the mentioned digital offset. Ultimately, this will not impact any experiment as the range of steering mirrors is typically several times larger than our typical DNA constructs (~ 1-2 µm). 46 3.1.3 Integrated shutter control Shutters are essential for trapping operations. To prevent trapping wrong particles and potentially having to restart the entire trapping process, the upstream trap is shut off via shutters, allowing the user to focus solely on getting particles into the downstream trap. Additionally, having the ability to remotely shut off the trapping beams reduces unnecessary noises from manual operation. Therefore, 2 independent shutters and controllers are installed within the beam-splitting stage (Figure 8-9, Chapter 2.2 â Stage 2 â Beam-Splitting and Steering). We have successfully integrated shutter control into the DAQ-SG program. Communication with the 2 shutter modules (SHB1T, Thorlabs) was also done via a data acquisition card. We used digital I/O ports to send a transistor-transistors logic (TTL) pulse 5V and 0V to modulate the shutter externally. (Figure 23) Figure 23. Shutters control mechanism and control scheme. (a) The function constantly assigns a DC signal of 0V (close) or 5V (open) to the DAQ card to output to the shutter controller. (b) The transistor-transistor logic in the shutter controller allows the user to modulate its state externally. (c) The user interface buttons for individual shutters. 3.2 Motorized stage control (MSC) The 3-axis stage (PT3A, Thorlabs) is driven by three high-resolution motorized servo actuators (Z825B, Thorlabs). Aside from the limited availability of commercial solutions, we determined servo motors as the most appropriate option since they offer better positioning accuracy than 47 their stepper counterparts. While both suffer from potential backlash compared to piezo actuators, the specialized servo offered by Thorlabs has a backlash of < 8 µm, which is negligible for our application since we typically reposition particles in hundreds of microns within the microfluidic chambers. Each actuator has a 25 mm travel range, enough to cover the multi-flow layout of our chambers. The actuators are controlled individually by controller modules (KDC101, Thorlabs). The controller offers local and computerized solutions with a physical interface with a velocity wheel and external USB communication port. Our program is built based on the integration package provided by Thorlabs via this communication. (Figure 24) Figure 24. Technical diagrams for the Main Stage Control (MSC) program. (a) three main states of the program and their corresponding features. (b) The program remains in the IDLE state until it is switched to the ACTIVE state upon a user trigger. It will stay in this active stage, continuing to send the signal to the motor controller (light blue) until the idle countdown is reached and no further commands are input. (c) The user interface of the MSC program features a digital limit specified by the user, different input methods and location memory. 48 Unlike the task-based nature of the DAQ-SG program, the Main Stage Controller (MSC) is reactive, and its default stage is idle. Once the program is turned on, it initializes all the parameters (hardware ID, communication port, default actuator velocity and acceleration, etc.). It stands by for an event generated by the user. This is typically a key press for stage translation or a change in values in the user interface (UI). Users can use the WASD key or enter the absolute position in all XYZ directions to command stage movement. The program is one big task that continuously adapts to user-specified commands. It is not feasible to state machine design for MSC. This is because, with user inputs, the number can be in the range of thousands during the experiment, while it demands the lowest response time to trap particles effectively, state switching is not feasible and unnecessary. Additionally, regarding the ability to operate and trap particles, the user sometimes requires multiple inputs (i.e., To move diagonally requires at least 2 inputs), making implementation even more difficult as the state must be subdivided to deal with each case separately. To prevent resource hogging, especially during force measurement when stage movement is not required, a temporary idle/shutoff mode has been implemented with an adjustable idle timer. One of the most immediate hazards while using MSC is crashing the chamber into the objectives, knocking them out of alignment and potentially damaging them. Along with the mechanical safety system, we have also implemented additional safety features that prevent the user from doing so. Range limit nodes are added for all XYZ axis controllers. Upon reaching the specified value, all further input from the user is rejected; only reverse movement is possible. The user can adjust this limit freely to adapt to minor variations between microfluidic chambers and different layout designs. On the other hand, basic input handling practice is important in ensuring better user experiences. Keys and switches in the UI that are not supposed to be pressed simultaneously, such as opposing movement keys up and down or left and right, would be greyed out and disabled if the user is already pressed on one of them. This acts as an indicators for the current input and prevents unwanted command execution. Lastly, a safety SHUT OFF button movement is implemented if the program is stuck in a translation move, forcing all controllers to stop all executions. 49 3.3 Particles tracking with CMOS camera CMOS sensors play an integral role not only in sample visualization but also in troubleshooting and alignment protocol. Thorlabs has provided a complete software package for live viewing, capturing, and analyzing images. While LabVIEW® integration packages are available, they lack basic features, such as on-screen crosshair, image crop and on-screen annotations. Hence, we used the provided software instead of developing a custom solution. Efficient active trapping operations necessitate a live video feed with a reasonable frame rate. This requirement arises from anticipating particle flows and positioning the sample stage accordingly, ensuring that traps are positioned ahead of the particle of interest. A lower frame rate poses challenges in particle detection and increases the risk of trapping multiple particles simultaneously. Based on our observations, a minimum frame rate of 30 fps is essential for achieving efficient trapping outcomes. The frame rate limitation is likely attributed to hardware constraints, with the data-streaming process of the DAQ-SG program being particularly resource intensive. Nevertheless, a potential solution to increase the frame rate involves sacrificing a portion of the field of view (FOV) by employing cropping or image binning techniques. (Figure 25) We prefer the binning approach, as it allows us to retain the full field of view necessary for particle detection while providing better positional awareness, preventing the traps from getting too close to the chamber walls or avoiding incoming beads. However, there are instances where the former approach is necessary, especially when our experiment requires enhanced image quality. Cropping to the trap regions allows us to record hundreds of frames per second. Nevertheless, at this FOV, attempting to trap new particles becomes extremely challenging. 50 Figure 25. The user interface of the CMOS sensor program. The left image shows a live view that has undergone binning (2x) result in a noticeably higher frame rate than a raw live view (right). White boxes highlight the essential features included in the program, which is used frequently for troubleshooting and trapping operations. Lastly, the dashed yellow box shows a cropped live view, which significantly boosts the frame rate of the raw live view. This method is often employed when the user wants to survey the particles closely during the experiment. 51 4 INSTRUMENT CHARACTERIZATION This chapter comprehensively explores various aspects of the instrument and presents fundamental information concerning the entire characterization process. Initially, we examine background noise levels originating from diverse sources, including mechanical and electrical, to assess the feasibility of mitigation strategies. Subsequently, we calibrate pertinent electrical components, such as the QPD, steering mirror, laser, and camera, to convert voltage analogs and digital samples into meaningful physical parameters for force and displacement analysis. A thorough evaluation of the instrument enables users to comprehend its capabilities fully and provides them with profound insights into data interpretation and potential troubleshooting aspects in the future. 4.1 Assessment of systemic noises Managing the inherent noise floor of the optical tweezers setup is crucial for achieving high spatial/force resolution. There are 2 main factors contributing to the apparent noise floor: environmental noise (discussed in section 2.6 â Instrument enclosure and hardware safety) and systemic noise. Systemic noise can be further broken down into 2 sources: laser noise from the laser module and electrical noise produced by the detection equipment. Noise minimization often comes down to careful planning of optical assembly, precise alignment, and implementing feedback control. In pursuit of ultra-high force resolution measurement, further improving the setup would be difficult without getting into custom-made components, advanced software solutions and complex optical assembly. Hence, it is necessary to assess each noise source individually to determine the optimization strategy that fits our system scope. 4.1.1 Laser noise Commonly, a solid state laser such as ND:Yag (1064nm) and Ti:sapphire (tuned 825nm) are used for trapping applications due to tighter line width and higher power output than typical diode lasers. However, they exhibit a certain amount of noise that could potentially impact the resolving power of the instrument. Laser noises consist primarily of intensity noise and mechanical noise. Since the laser is monochromatic, the effect of frequency noise is minimal and, thus will not be assessed in this section. 52 Intensity noise is the fluctuation of laser power over time. It is the direct result of laser design and manufacturing efforts. It can be reduced with thermal management, component quality, and a stabilization strategy. The instability of the laser power causes a shift in trapping potential, which allows for additional random motion of trapped particles. Additionally, with the dumbbell trap array formed from a single laser, fluctuation in laser power may affect individual traps differently, further complicating calibration effort. Good laser practices have been implemented, from setting the laser at high power (80%), placing the laser module in a well-ventilated, temperature-controlled area, and having a Faraday isolator and polarizer to increase its lifetime. However, assessing the degree of noise exhibited by our laser module is still necessary, as it can potentially be a troubleshooting solution for any trapping issues raised in the testing phase. We assessed the laser output during the first hour of operation. (Figure 26) Figure 26. Laser power measurement during the first hour of operation. Power fluctuation is measured using a QPD sensor at the end of the optical train, where A and B represent the fixed trap and steering trap, respectively. It can be characterized into three categories. The laser module warms up to equilibrium, where its components undergo internal thermal management. This results in a significant voltage swing (red segment). Instrument optics absorb some irradiation power and heat up, resulting in a slight drop in measured power (yellow segment). Lastly, no further noticeable changes (blue segment) signifying the entire system is fully heated and ready to be used. The laser power exhibits significant fluctuations during the initial 45 minutes but stabilizes to less than a 1.5% variation after one hour of heating. Beyond this point, the system reaches equilibrium and becomes operational. It was observed that any attempt at data collection 53 without finishing the warm-up process would result in skewed data and, often enough, inaccurate force measurement. This is due to the inconsistency in trap stiffness due to the drastic changes in laser power, as observed in Figure 26. Managing the fluctuations further would entail evaluating the cooling solution of the laser module and controlling the ambient temperature. Nevertheless, both measures would require a substantial investment of time and effort. These considerations are noted for future improvements should the need for higher-resolution applications arise. Finally, we assess the pointing stability by measuring the position of both lasers without trapping any beads. The assessment is done after the system is built and calibrated. Figure 27. Laser-pointing stability assessment. QPD sensors record the X and Y displacement of each beam (A â fixed trap and B- steering trap) after being centred and fully warmed up. A microfluidic circuit with no particles was installed to replicate the experimental condition closely before the measurement was made. The displacements are offset by 10 nm from one another for display. In Figure 27, the pointing stability varies up to 2.5 nm over 15 minutes. This is on par or better than the reported thermal drift of other systems where severe drifts as high as 2 μm/min were observed. 107 It was observed that there is a drift for beam A in the Y direction. Additionally, noise levels in both directions for all traps appear unidentical. The displacement signals fluctuate on an average of 2 nm, meaning the instrument can resolve force 54 measurements of up to 1.2 pN. This issue will most likely be resolved by better mechanical noise isolation strategies such as sample stage isolation, optical cable dampening, etc. Ultimately, the noise floor needs to be reduced by at least half to achieve sub-piconewton force resolution. To do so, extensive room treatment or relocation of the instrument to a dedicated basement area with regulated temperature and airflow is required. 4.1.2 Electrical and mechanical Like any sensitive measuring instrument, optical tweezers are susceptible to electrical noise. Typically, the noise comes from the detection components, where sensor readings are collected and stored digitally. Voltage fluctuation (ground loops) may occur as electrical components have separate grounds. We first assess the noise level of the data acquisition card using our data collection program, which involves connecting all cables and turning off all other components. Then, we record noise measurement of other components individually, namely the piezo steering module consisting of the controller along the piezo unit and the QPDs with their power supplies. Electrical noise resulted in ~0.012 nm in positional uncertainties, an insignificant amount as compared to the mechanical noise (Figure 28) Figure 28. Electrical noise measurement for different states of the instrument. The data acquisition recorded the voltage reading of a QPD sensor (DAQ) card. When the system is off, the noise consists of inherent noise from the DAQ card and the computer, which has a separate grounding (red segment). The noise level observed when the system is turned on is slightly lower, possibly because the 55 component now shares a common ground. When the laser is turned on, laser pointing stability becomes the major contributor to background noise (blue segment). The mechanical instability exhibited when the system is powered on stems from not only the laser instability mentioned in the previous section but also the accumulation of vibrations from the external environment, such as the location of the instrument, incomplete isolation of cables and the inability to control airflow within the instrument room fully. Finally, the impact of electrical noise on frequency-domain analysis, such as power spectrum density (represented by a spike at 50-60 Hz), is shown to be minimal in the subsequent calibration chapter 4.2.1, Figure 30. 4.1.3 Steering mirror linearity and conjugation The moving trap was created by a piezoelectric motor (Newport, PSM2SG), controlled by a piezo stack amplifier (Newport, NPC3SG). While the piezo element has several advantages over stepper and servo motors, such as having no backlash and higher precision, it suffers from lagged movement, also known as hysteresis. To combat this problem, the positional feedback system is usually implemented to ensure the mirror mount moves according to the applied voltage. To assess the steering performance, we directly compare our drive signal with the monitor signal received from the controller. 56 Figure 29. Linearity and timing assessment of the piezo steering motor. (a) The voltage signal assigned to drive the motor and the feedback signal were recorded at 1kHz, showing a peak mismatch of 5 milliseconds. (b) With closed-loop feedback control, the steering mirror exhibits minimal hysteresis. However, the non-overlapping (small inset) between the drive and monitor signal can be attributed to the timing mismatch mentioned previously. This is not a dynamic lag caused by hysteresis since the response is linear, which is expected as the controller constantly compensates in closed-loop feedback. A simple hardware delay explains the mismatch between the peaks of the two signals. A delay of 5ms (Figure 29a, 57 inset) is calculated from the number of data points mismatch between the steering feedback and the actual QPD signal. This observation is further reinforced by the presence of a small gap in the second plot (Figure 29b, inset), indicating the presence of a timing mismatch between the two signals. Because the delay is consistent between different steering amplitude and steering frequencies, a hard-coded data offset of 5ms is included within our analysis pipeline. 4.2 Instrument components calibration This section discusses the calibration of all electrical components involved in force measurement. Each component by itself produces information that is used as a building block for the Hookean linear spring model: ð¹ð¹ = âðððð (9) Where ð¹ð¹ is the force applied, ðð is the spring constant or trap stiffness, and ðð is the spring extension. Since back-focal plane interferometry is used to gather positional information of the trapped particle, it utilizes the QPD to collect light as it passes through the optical train. Hence, the technique is sensitive to not only the morphology of the particle but also external local changes within the system (thermal/mechanical noise, microfluidic flows, etc.). System calibration is required for each pair of trapped particles, and therefore, equation (9) can be written as: ð¹ð¹ = âðððððð (10) To get meaningful parameters such as force and the extension of the molecule, conversion factors such as detector sensitivity or optical sensitivity ðð and trap stiffness ðð need to be determined. As the displacement signal ðð is measured in analog voltage, it can be converted to physical extension ðð using the factor ðð (V/nm). Once the extension is calculated, the applied force is evaluated using the trap stiffness ðð (pN/nm). Therefore, besides the one-time calibration of the camera system ð¶ð¶ and the steering mirror factor ðð, individual calibrations of ðð and ðð are always accompanied by every single dataset to make sense of force measurement. 58 4.2.1 Trap stiffness ðð - Power Spectral Density Optical analysis is one of the two requirements for absolute force measurement. It characterizes the trapping force and allows the conversion of the particle displacement voltage signal to force units. This conversion factor - also known as trap stiffness ðð - is highly sensitive to the particle sphericity, density and material quality. As such, it varies noticeably from one particle to another. Hence, the calibration must be done routinely for every trapped particle. There are several ways to perform trap stiffness calibration, of which the power density spectrum analysis is the standard method of choice due to its reliability and ease of use. In addition, the analysis is done on the frequency domain and, therefore, is not subjected to the influence of low-frequency thermo-mechanical noise within the system. An optically trapped particle agitates within the trapâs harmonic potential due to the thermal fluctuations (Brownian motion) and displacement from the trapping centre. As the particle is confined within the trapping region, the motion of the particle is suppressed and exhibits sub-diffusion behaviour. Typically, the particle oscillates about the equilibrium point at a frequency of several kiloHertz. Therefore, the XY positional voltage of the particle is recorded at 100kHz sampling frequency to capture particle motion fully. The experimentally determined power spectrum of the particle displacement carries a Lorentzian shape and is the result of the time-Fourier transform for the square of displacement ðð: ððð¥ð¥(ðð) = ðððµðµðððð2ð¾ð¾(ðð2 + ððð ð 2) (11) where ðð(ðð) is the power spectrum showing the distribution of noise power over the corresponding frequency, ðððµðµ is the Boltzmann constant, ðð is the temperature, and ð¾ð¾ is the hydrodynamic drag on the probe particle. For a low Reynolds number, the hydrodynamic drag coefficient ð¾ð¾ is determined as: ð¾ð¾ = 6ððððð ð (12) with ðð being the dynamic viscosity of the trapping media (it is water in our case) and ð ð is the radius of the particle. The characteristic roll-off frequency ððð ð is related to trap stiffness ðð given by: 59 ððð ð = ðð2ððð¾ð¾ (13) The power spectrum can be divided into 2 regimes by the roll-off frequency ððð ð . The low-frequency region (ðð âª ððð ð ) is approximately plateau as it represents the suppressed oscillation of the particle. At high frequency (ðð â« ððð ð ) region, the power spectrum drops as 1/ðð2, showing slope -2 in the log-log scale (Figure 30, green region). This fall-off indicates free diffusion of the particle within the trap where the displacement ðð of the particle is smaller than the trap diameter. This behaviour can be mathematically described as:108 ðð âª ððð ð â ððð¥ð¥(ðð) â ðð0 = ðððµðµððð¾ð¾ðð2ððð ð 2 = (2ðððððð)ððððððð ð , (14) ðð â« ððð ð â ððð¥ð¥(ðð) = ðððµðµððð¾ð¾ðð2ðð2 â 1ðð2 (15) Figure 30. Power spectral density plot of optically trapped particle. The vertical lines indicate the corner frequency ðððð for each power spectrum. The green region is where ðð â« ðððð while the orange region represents ðð âª ðððð 60 Stiffness calibration also reveals imperfection in the construction of the instrument, specifically in the alignment of all optical components. As trapping power is proportional to the laser power, a well-aligned instrument demonstrated this linear relationship clearly if a particle is trapped and calibrated at a series of increasing laser power. (Figure 31). Furthermore, a histogram of the displacement distribution of a calibrated particle can be generated from the calibration dataset to investigate the beam profile received by the QPD at the end of the optical assembly further. Any clipping of the beam of off-angle alignment will result in asymmetrical or misshaped distribution. Overall, the system is well-aligned, as shown by the stiffness response to laser power and the expected bell-shaped distribution of the trapped particle position. Figure 31. Trap characterization. (a) Trap stiffness is a power function; a linear relationship is expected if the instrument is well aligned. The inset shows a residual plot with power fluctuation within ±0.01 pN/nm. (b) Normalized histogram plot for the displacement of a trapped particle at 800 mW for 30 seconds. The distribution (blue) is well matched with the bell curve (red) representing the intensity profile of the Gaussian beam. 61 4.2.2 Optical sensitivity ðð â Flow calibration Optical sensitivity ðð refers to the ability of the detectors to measure the positions of a trapped particle. As the particle moves away from the trap center, the detector voltage increases linearly until it reaches the escape threshold. Through calibration, this voltage is transformed into the trapped particlesâ physical displacement, revealing the conformational dynamic of the tethered molecule of interest. The first approach directly correlates the voltage to the particle's distance from the centre. To achieve this, a specially treated cover glass surface is necessary to facilitate particle adhesion. A factory-calibrated stage motor is programmed to perform a sweep or sinusoidal motion signal, causing the particle to move across the trap. The QPD's voltage (ððððððð·ð·) is then monitored to establish a ratio between the actual distance travelled (ð·ð·ð ð ððð ð ð£ð£ðððððððððð) by the particle and the corresponding voltage reading (V/nm). â = ð·ð·ð ð ððð ð ð£ð£ððððððððððððððððð·ð· (16) With the second approach, we correlate the drag force experienced by the particles with the voltage changes. (Figure 32) A trapped particle was put under a ramping flow series. The drag force of this particle in a microfluidic environment (low Reynold number) can be calculated as: ð¹ð¹ððððð ð ðð = 6ððððð ð ðð (17) Where ðð is the dynamic viscosity of the fluid, ð ð is the radius of the particle, and ðð represents the flow velocity experienced by the particle. Flow velocity is dependent on internal channel geometry: ðð = ððð´ð´ (18) Q is the current flow rate, and A is the cross-sectional area of the chamber. Under flow, the particle will be pushed away from the trap centre, leading to a voltage difference in the QPD quadrants. Thus, the relationship between it and the corresponding calculated drag force can be established (V/pN), and by performing trap stiffness calibration (pN/nm), we can deduce the optical sensitivity of the system (V/nm). 62 â = ð¹ð¹ððððð ð ððððððððð·ð·Ã ðð (19) This approach necessitates minimal preparation, including surface treatment, adhesion protocol, and specific bead types, among other factors, and can be conducted in conjunction with routine experimentation. As a result, it is favoured over its predecessor. We calculated our optical sensitivity to be 100 nm/V. Figure 32. Determination of optical sensitivity ðð by flow series assay. A calibrated particle is optically trapped and undergoes ramping flows of the experimental buffer. As the flow rate increases, the particle will be increasingly displaced from the trap centre (left scheme). The displacement is recorded by the QPD sensor. The drag force from each flow rate is calculated and compared to the corresponding displacement from the measurement (right plot). A ratio force and voltage displacement can be established, which is finally converted to optical sensitivity ðð (in V/nm) from the determined stiffness of the trapped particle. 4.2.3 Pixel size ðªðª â Camera calibration The camera is an essential aspect of the system thanks to its ability to visualize the trapping plane. It enables live monitoring of the traps, prevents trapping multiple particles and, most importantly, navigates flow channel topography. The calibration of the camera allows the user to be aware of the relative position between the traps, which is required for the subsequent steering mirror calibration in the next section. Particles were flowed into the chamber and allowed to stick on the functionalized surface of the channel. This process can be sped up by trapping a particle and making contact with the 63 surface. The particle stuck on the surface is then used as a reference point for us to perform pixel calibration using predetermined stage translations. The d isplacement of the reference particle ð·ð·ððð ð ððð ð ððð ð ðððð can be compared to the distance moved via the stage ð·ð·ð ð ð ð ð ð ðððð to determine the pixel-to-distance ratio, also known as pixel size: ð¶ð¶ = ð·ð·ððð ð ððð ð ððð ð ððððð·ð·ð ð ð ð ð ð ðððð (20) with ð·ð·ððð ð ððð ð ððð ð ðððð is measured in pixels and ð·ð·ð ð ð ð ð ð ðððð is reported by the stage translation motor in microns. For accuracy assurance, measurements were collected across multiple distances. The camera view also experiences a wrapping effect near the edge of the sensor, which causes the pixel size to vary noticeably. Nevertheless, the trapping operation only happens over a small area at the very centre of the camera sensor, where the pixel size ð¶ð¶ is found to be consistent at 1 µm/pixel. (Figure 33) Figure 33. Schematic for pixel size ðªðª determination method. A stuck particle is focused on the live view of the CMOS camera. The stage is translated by a pre-determined amount using a calibrated stepper motor. Its position is captured as a series of images during this process. The pixel displacement of the particle is directly linked to the translation performed, and thus, the pixel size can be determined. Multiple runs were performed in each X Y translation, and the result is average to account for translation motor repeatability and mounting stage error. 4.2.4 Steering sensitivity ðºðº â Steering mirror calibration During tethering experimentation, assigning a signal to the mobile trap requires the operator to know the exact voltage conversion in order to avoid the tether from potentially being overstretched or completely broken off. Furthermore, voltage conversion is also needed to calculate tether extension. To perform this calibration, a particle is trapped using the steerable trap. The steering trap is then moved horizontally in 1V intervals. The particleâs visual position is recorded after each 64 move via live imaging. With pixel size C determined, the actual distance corresponding to each voltage can be determined, and voltage conversion is calculated: ðð = ð·ð·ð ð ð ð ððððððððð ð ð ð ððððððâ ð¶ð¶ (21) Where ðð is the steering sensitivity of the piezo motor, ð·ð·ð ð ð ð ðððððð is the displacement of the particle as the moving trap is steered, ððð ð ð ð ðððððð is the amount of voltage applied to the motor to steer the trap. As demonstrated in Figure 34, the process is repeated for different voltage intervals (1V, 2V, 5V, 10V), and different directionality (left-right, up-down and vice versa). With closed loop mode, our steering sensitivity S is 748 nm/V in horizontal and vertical directions. Figure 34. Schematic for determination of steering sensitivity ðºðº. A particle is optically trapped using a steering trap. Its position is recorded using a calibrated CMOS sensor mentioned in the previous section, 4.2.3. The particle is then steered for a pre-determined voltage (1-5V). Since the pixel size ðªðª is known, a relationship between steering voltage and actual particle displacement (in V/nm) can be established. To cross-validate this result, another protocol is established that does not rely on the pixel size calculation. This method relies on our stiffness calibration to determine the drag force applied to the particle and the speed at which it travels. A particle is trapped and steered under 65 different rates under a triangular waveform. The position of the particle is measured at a sampling rate of 10kHz. From power spectrum analysis, we determined the stiffness ðð of the trap on the target particle in both X and Y directions. We can then calculate the drag force ð¹ð¹ððððð ð ðð being applied on the particle during high-speed steering: ð¹ð¹ððððð ð ðð = ððððððð·ð· â ðð (22) where ðððððððð is the voltage measurement of the QPD reporting the instantaneous position of the particle during the steering process. The steering velocity ððð ð ð ð ðððððð is then evaluated: ððð ð ð ð ðððððð = âððð ð ð ð ððððððâð¡ð¡ (23) where ð¡ð¡ is the sampling period. By comparing the rate of change in drag force as velocity increases, it is possible to determine the steering sensitivity: ð¹ð¹ððððð ð ððððð ð ð ð ðððððð= ð¹ð¹ððððð ð ðððððððððððð= 6ððððð ð (24) â ðð = ððððððððððððð ð ð ð ðððððð (25) with flow velocity ðððððððððð in nm/s and the ððð ð ð ð ðððððð is in V/s. We have achieved a relatively similar steering sensitivity of 751 nm/V to the prior method. More importantly, this cross-validation technique also confirms the validity of the camera calibration stiffness calibration. With all the calibration factors determined, we can convert all the voltage data into meaningful values. For example, a particle is displaced from the trapping centre, resulting in a 0.7 V change in the detection signal. Using trap stiffness ðð (0.6 pN/nm) and optical sensitivity ðð (100 nm/V), we can deduce that the particle is experiencing a force of 42 pN and moved 70 nm off the centre of the trap. The calculated force measurement can then be used to construct the force-extension curve, fit it to the appropriate model, and validate it with literature values. 66 5 PROTOCOL FOR SINGLE-MOLECULE EXPERIMENT This chapter outlines the development of custom flow cell construction and material preparation tailored for typical single-molecule experiments, specifically for our optical systems. The flow cell design for force-measuring optical tweezers must adhere to several physical constraints, which will be discussed later. It is important to note that optical tweezers are frequently home-built, rendering these constraints inapplicable to all setups. Nevertheless, the primary aim of this chapter is to provide a general understanding of custom flow cells along with considerations and design guidelines. We have successfully established a robust fabrication protocol that is both user-friendly and time-efficient. In addition, we report a robust and thorough particle preparation strategy that resulted in a high homogeneity level, facilitating a smooth and efficient trapping operation. 5.1 Multi-flow chamber fabrication Optical tweezer experiment typically uses a microfluidic chamber as the trapping environment. These chambers are often made using two pieces of a glass slide with a spacer in between. Thin glass reduces optical aberration from media mismatch; they are typically 100-150 µm thick and can be purchased commercially through various vendors. We source our glass slides via Fisher Scientific (Premium Cover Glass Type 1, 12-548A-5PP); the cover glass is about 130 µm thick. The spacer, arguably the most important piece of the chamber, functions as the internal layout and a binder between the 2 glass slides. Different channel geometry can be achieved for any custom application, depending on the spacer design. Therefore, these sample chambers are fabricated in-house for individual optical tweezer setups. Spacers can be made of Polydimethylsiloxane (PDMS)98,109 or wax film such as parafilm23,110. PDMS film creates a strong chamber that can withstand a high flow rate. Moreover, PDMS enables detailed and intricate channel designs with high consistency. The biggest drawbacks of PDMS moulding are the cost and fabrication difficulty. The moulding mask is costly and usually has to be outsourced, while the curing and chamber fabrication process requires training effort and specialized equipment. On the other hand, 67 parafilm chambers are inexpensive and easy to fabricate. While chamber quality is not as consistent as the PDMS spacer, the tolerance is acceptable for simple microfluidic channel design since our stage motion is on a micrometre scale. Hence, we developed our own parafilm flow cell fabrication protocol. The chamber must satisfy several general criteria: 1. Thickness: As thin as 50 µm, the channel depth negatively impacts trap performance. Due to decreased channel volume, it is also more accessible for the user to find and track beads. On the other hand, if the channel is too thin, additional hydrodynamic effects should be considered due to the particleâs proximity to the surface. 2. Multi-flow design: It is possible to have a single flow of pre-mix materials such as pre-tethered DNA âdumbbellsâ. Finding and trapping a single pair could be a challenge. Therefore, it is better to have separate flows containing each type of particle than to trap each component individually. 3. Complete spacer sealant: The Spacer, in our case, parafilm, must be evenly sealed such that there are no trapped air bubbles or weak binding spots that could be visually detected. This reduces the chance of channel leakage and air bubbles being introduced into the flow channel during long hours of experimentation. 4. Reusability: After multiple uses, the spacer materialâs seal and the top coverglass's holes must remain intact. Achieving this criterion will reduce subsequent preparation time and waste. We achieved this protocol set by adapting and modifying protocols from existing literature.23,111 5.1.1 Coverglass preparation Two pieces of identical-sized coverglass are used to construct a microfluidic chamber. A set of predetermined holes is drilled into one of the cover glasses to serve as openings that allow the introduction of liquids into and out of the internal channel. The holes on this coverglass must be no bigger than 0.5mm and have no cracks within the proximity. This ensures optimal mating with the tubing of the inlet. Manually drilling the holes using a rotary motor is often time-consuming, while the delicate coverglass has a high tendency to break. The laser cutter 68 (LS-1420, BOSS LASER) produces the most consistent and precise holes on coverglass. A custom holder tool is developed along with an optimized laser drilling tool path, and thermal control solutions enable mass production with minimal oversight. After drilling, the coverglasses are sonicated under ethanol and 1M KOH solution. Then, a final rigorous wash with MilliQ water is needed to remove any remaining glass impurities completely. As mentioned in the prelude, chamber surface preparation plays an important role in the longevity of the chamber. Particles flowing within the channels might get stuck to the surfaces, and accumulations of stuck particles potentially reduce the quality and efficiency of the trap due to unwanted scattering and absorption of these stuck particles. Additionally, they may negatively influence the channelâs altering flow rate. Some may get dislodged as large clumps and impede the trapping operation. To prevent this problem, the internal surface should be passivated. Passivation can be done by incubation beforehand using common blocker proteins such as BSA112, casein113, etc. or polymers such as PEG114, Poly-L-Lysine115 and tween-20116. We chose the BSA approach due to its simplistic procedure and optionally passivate with Tween-20 should the need for a better-quality surface arise. The passivated chamber shows a reduction of stuck beads by ~90% based on qualitative visual assessment. The chamber can be reused up to 3 times before parafilm integrity is compromised. It should be drained and washed with MilliQ water, followed with ethanol, and incubated with fresh 10% sodium hypochlorite for 5 minutes. After incubation, the chamber is triple-washed with MilliQ water. Each wash was done by injecting the washing solution 200 µl through the outlet of the assembled chamber and actively removing the solution on the other side using a piece of Kimwipe paper. Lastly, the chamber can be fully drained by blowing clean air (in our case, N2 gas) into outlets. Once dried, it can then be stored in a sealed slide container, ready to be used for the subsequent experiment. 5.1.2 Precision-cut channel layout In contrast to the complicated PDMS curing and moulding process, parafilm is not only safe to handle since it is chemically inert but also very flexible in terms of instrument use. It can be moulded or cut using a laser or sharp tool. Initially, we use a laser CNC, which allows high-precision cuts with sub-mm resolution. We quickly discovered that laser heat melts the parafilm, creating an inconsistent surface that might negatively impact the subsequent chamber 69 sealing process. Moreover, the internal channel edges become jagged, thus allowing particles and air bubbles to stick along the wall of the channel, affecting the overall performance of the chamber. This is the result of the high-speed passing of the laser cutter, which is unfortunately unavoidable since parafilm has a low melting temperature (65º C), any low-speed cutting attempt results in a deformed layout and curling. Additionally, the optimization process is extremely involved. The tool path needs to be cut in a specific way to avoid the overhang portion, which may result in an incomplete cut. Additionally, temperature control via cutting speed and cooling fan prevents deformation. Vibration reduction is critical due to the elastic nature of the parafilm and the intricate channel design. Therefore, we abandoned this approach. Utilizing a computer numerical control (CNC) router allows us to save time and scale up the production of the chambers instead of manually cutting the layout. However, since the laser cutter approach is challenging, we switched to a mechanical router. With light applications involving cutting tape and parafilm, commercial consumer-grade machines such as Cricut® Maker is more than enough for our needs. The parafilm is cut into strips of 50x25 mm and loaded onto the cutting mat. After running through the CNC router, they are gently cleaned using pressurized clean air and ready to be installed on the cover glass. With this setup, we achieve the layout with a resolution of hundreds of microns while maintaining the smooth channel edge. Additionally, no other custom tool or cooling is required, further simplifying the fabrication process. Under a microfluidic environment, liquids do not mix and exhibit laminar flow behaviour. Therefore, it is possible to introduce multiple flows of liquids, each containing a population of different particles, into a single channel. This allows us to have better control of tether formation than the pre-mixing particles method. The channel layout allows three different liquids to enter a single channel. The two top and bottom channels are merged into the middle channel at a small 15 degrees to smoothen the flow. All sharp corners are rounded to minimize any potential turbulence. The connections are typically 100 microns wide, although this may vary from channel to channel due to human error. It is recommended that narrower connections be made to concentrate the particles, which improves particle detection and tracking at the junction. We also pushed the junction where particle tracking and trapping are slightly 70 downstream. This enables us to access some upstream portion of the middle where no particles are present. This region is perfect for initiating measurements and experimentation since there will be no disruption from random particle trapping. (Figure 35) Figure 35. Flow cell anatomy and operation. Two coverglass sandwiched a custom-cut parafilm with the microfluidic layout, allowing multiple flows of buffers to merge into a single channel. The top coverslip is drilled with openings such that 2 different particle solutions and an experimental buffer can be simultaneously introduced. Typically, particles are trapped at the channel junction where both top and bottom flows are accessible. Once the particles occupy the traps, they are repositioned to the upstream part of the middle channel to avoid accidental trapping of additional particles (small inset). 5.1.3 Fabrication protocol The components are put together in a clean, dust-free environment. We used a vertical laminar flow hood to store and assemble our chamber. The drilled coverglass is placed on a clean mat, followed by the parafilm layout placement, and another coverglass is placed on top. Assembling in this sequence ensures proper layout alignment with the drilled holes. Once the 71 components are placed, a gentle but firm force is applied across the chip using a pipette tip to bind all the components together. After trimming excess parafilm, the chip can be fully sealed using a hot plate or the oven. The assembled chip was placed directly on a hot plate preheated to 90 degrees Celsius; then, an aluminum block was placed on top to produce even heating. The chip was left on the hotplate for 5 minutes before being flipped upside down and heated for an equal amount of time. From our observation, the hot plate method often produces sub-par quality chips: an incomplete seal that traps air bubbles, eventually leaking into the flow channel or entirely separating the coverglass. (Figure 36) Often, a good indication of a bad seal can be seen with stuttered particle flow, sometimes even backwards. We switched to using an oven to seal the chip thermally. While this method requires precise timing and preparation, the resulting chips are significantly better and reproducible. The assembled chip is tightly clamped between 2 glass slides and heated up to 75 oC for 1 minute. Then, the clamping contraption is removed, and the chip is transferred to another oven that heats it to 120 oC for another minute. The first stage, the âpre-sealingâ stage, is crucial as it removes all trapped air within the parafilm interface. The subsequent sealing stage ensures the parafilm spacer completely melts and binds two chamber coverglass surfaces fully. The timing for each stage may vary depending on the type of glass used. Stage optimization is needed to ensure not only the seal quality but also the flow layout integrity. Overdoing any of the 2 mentioned stages, especially the initial âpre-sealingâ stage, would lead to rapid deformation of the channel layout. Generally, the thicker the glass used for the chip, the longer the âpre-sealingâ stage needs to be from 1 minute (#1 coverglass, with a thickness of 130-170 µm) up to 2 minutes (glass slide). On the other hand, the second stage has more leeway. The chip can be heated for a maximum 2 minutes regardless of the glass thickness. Any extended heating will result in an inward expansion of parafilm, covering up the channel layout and the chamber holes. 72 Figure 36. Visual comparison between different parafilm sealing techniques. A hotplate is often employed to seal the parafilm layer between the glasses (top image). We developed a new sealing technique using a two-stage process with initial binding (75 oC) and final seal (120 oC). This technique produces a more consistent seal and minimizes channel deformation (bottom image). 5.2 Nanoparticles preparation A successful optical trap experiment heavily depends on the particle solution's homogeneity. Particle clumps, especially as pairs, could be tough to differentiate from single particles as they usually appear as a single particle on camera, with one particle blocking the other. Additionally, a mishandling particle solution would result in a noticeably small percentage of single particles, creating challenges for users to find and track. For bead functionalization, 73 inconsistent surface coating due to aggregation potentially leads to extended tethering formation time and effort, limiting data output. Therefore, it is empirical to have a robust protocol that has high compatibility with a wide variety of particles. 5.2.1 Nanoparticle washing and handling protocol For trapping applications, the particles are usually microspheres with a refractive index higher than surrounding media to maintain enough refractive force and reflective force, which locks it in space. The particles should be equal to or larger than the wavelength of the laser (1064nm) so that ray optics is applicable. We used 1 µm nominal-sized particles (Streptavidin microsphere - CP01004, Protein A microsphere - CP02000) made by Bang Lab, IN. The new particles, which are larger, have increased our force range to a max force of 100 pN. Particle material is dependent on availability and popularity in the commercial market. Typically, polystyrene is used as the base material to manufacture microspheres thanks to its high refractive index (RI 1.59) while retaining optically clear properties. Conveniently, polystyrene microspheres also readily enable proteins and surfactants to adsorb thanks to its hydrophobic surface. Thanks to these advantages, polystyrene particle is often the option with the most diverse catalogue among vendors and thus used by most single-molecule labs. Before usage, particles must be washed and undergo a de-aggregation protocol to achieve high homogeneity. Typically, an experiment requires tens of µL of 0.001% w/v beads solution. We therefore prepare a stock solution for each particle type to be stored in storage solutions, typically TE (Tris 10mM and EDTA 1mM) or T50M5 (Tris 100 mM, NaCl 50 mM, MgCl2 5 mM) buffer depending on the presence of DNA along with the particles. The particle solutions are pipetted into a 500 µL micro-centrifuge tube and washed using a centrifuge twice at 10000 x g for 5 minutes. The storage buffer is replaced with 200 µL of running buffers. Finally, particles are sonicated for at least 60 seconds in a sonication bath. At this point, they are ready to be functionalized with either DNA or appropriate ligands. 74 Figure 37. Before and after images of particle stock after undergoing de-aggregation protocol. The 1μm-sized anti-digoxigenin coated particles are imaged using an IX83 microscope at 100x magnification under a bright field. Smaller particles tend to aggregate due to having a higher surface-area-to-volume ratio. This issue can be alleviated, as shown in Figure 37, with some good handling practices: (1) Mixing: Mixing particles between washing steps or during the transfer of particles should be done diligently. Both vortex and pipette mixing are recommended. (2) Micro-centrifuge tube: Tube size matters when it comes to sonication. We found that a 500 µL tube offers the best sonication efficiency. Larger tubes may dissipate too much acoustic energy, which reduces particle agitation. (3) Sonication volume: The sample volume must not be too large, such that acoustic energy will be dissipated as it propagates through the sample container, resulting in a lower density of cavitation bubbles, hence less agitation. On the other hand, a small sample volume may result in overheating of the particle solution, risking permanent thermal damage to the coated proteins. Experimentally, we find that 200 µL sample volume provides a good balance between sonication efficiency and sample heating. (4) Sonication time: Sonication time affects the degree of aggregations. Sonication time of 30 to a maximum of 60 seconds would result in a highly homogenous sample. Extended sonication may result in damaged particles and the removal of proteins from the coated surface. This concern can be partially alleviated if the proteins are covalently bound to the particle. 75 5.2.2 Nanoparticles functionalization protocol Choices of protein-ligand interaction dictate how efficiently the experiment can be performed. Depending on the tether construct, several specific interactions such as avidin-biotin or (anti)digoxygenin may be utilizedâcomplete constructs (ligand-tether-ligand) benefit from having different interactions at each end to prevent aggregation. The multi-part construct (ligand-tether[1] and tether[2]- ligand) simplified the system by requiring a single interaction, as the formation is done between the tether parts, as opposed to the protein-ligand interface. One of the most common approaches is forming individual dumbbells in situ (within the flow chamber). This is done by attaching the complete tether construct on one bead, usually with avidin-biotin interactions and bringing the other bead to proximity until the other end of the tether is attached to the free bead (hence the name dumbbell traps or dumbbell assay). While it is possible to pre-form dumbbells by mixing the two beads population prior to loading them into the microfluidic chamber117, controlled tether formation eliminates the effort needed to optimize the pre-mixing procedure, which could be challenging and inconsistent due to a variety of factors such as bead concentration variation, temperature, beads ratio and mixing volume. We first make our testing tether based on Woodsideâs hairpin design32. The DNA hairpin is called 15R60/T30 (15bp stem, GC 60%, 30Ts loop), with each end consisting of one biotin or digoxygenin molecule. The method of construction can be found in the appendix. We attached the testing tether onto SA particles and functionalized protein-A particles with anti-digoxygenin for our dumbbell system. These 2 particles will be used for the tether formation experiment. To make DNA coated streptavidin (SA-DNA) particles, 10 ng of 15R60/T30 DNA hairpin was mixed with 10 µL of stock SA particle solution to instigate binding via avidin-biotin interaction. Then, the solution is left for half an hour under room temperature conditions before it is made up to the final 100 µL stock volume using filtered TE buffer. To ensure proper mixing, the DNA solution is added in a small portion with aggressive mixing between each addition. This ensures all SA particles contain at least one DNA hairpin molecule, increasing trapping efficiency. For AD particles, a similar protocol was done using 10 µL solution of 76 protein-A particles is mixed with 50 µL of 150U stock anti-digoxygenin solution in T50M5 buffer. Both stock bead solutions are then stored in 4º C fridge. 5.3 Flow circuit assembly protocol About 10 µL of each bead solution was washed with T50M5 buffer using the wash protocol mentioned in section 5.2.1. Meanwhile, the fabricated chip is installed on a custom C-holder with drilled threads corresponding to the number of opening holes on the microfluidic chip. The chip should be installed with its holes facing toward the threads so that they are aligned. Then, two acrylic blocks should be used to clamp the chip firmly against the holder, preventing it from sliding around, while over-tightening may result in cracks and leakage. Subsequently, special inlets made of hollowed set screws that have tubing threaded through are used to direct the flow of experimental solutions into the appropriate channels within the multi-flow chamber. They are screwed into the threads of the holder, which are aligned with their corresponding opening holes of the chip. Some adjustments to the chip position may be needed. This can be done by temporarily loosening the clamping blocks and manually moving the chip to align the holes with the tubing of the inlets. A leakage test can be performed upon chip installation to ensure proper sealing. This can be done by pipetting 150 µL of running buffer and filling up the entire chambers and the inlets. Excess buffer forms droplets on top of all inlets. If the droplets remain for several minutes, the setup is considered leak-free. On the other hand, droplets gradually withdrawn into the inlets indicate leakage within the system. A troubleshooting table is included in the appendix to help identify the problem and propose possible remedies. Additionally, this test's secondary objective is to eliminate any bubbles trapped within the chamber. This is a critical step as they are incredibly disruptive during the trapping and data collection. Agitating the flows of running buffers by repeatedly dispensing and withdrawing is the recommended course of action. A final buffer consists of 50 mM Tris, 200 mM KCl (pH 7) buffer along with 2% protocatechuic acid/ protocatechuate-3,4-dioxygenase (PCA/PCD) oxygen scavenging buffer (10 mM PCA and 26 nM PCD, filtered) is used to make up 500 µL sample volume for each of the three samples: SA-DNA particles, No beads, AD particles. Traditionally, glucose oxidase 77 or pyranose oxidase and catalase (also known as GODCAT) are used to remove oxygen from the running buffer. This is a cheap and efficient system with 2 major drawbacks. Firstly, it has impurities ranging from 100 nm to 20 µm, mostly from catalase. Despite our best filtration efforts, namely centrifugation and sample filtration, we could not remove the sub-micron impurity. Secondly, acidification due to gluconic acid production could potentially damage biological samples. PCA/PCD was therefore used to avoid these problems and achieve lower O2 steady-state concentration. 118 Then, all samples are loaded onto perspective syringes (Hamilton, 1650LT 500ul SYR) using long needles (BD, 305165). Bubbles accidentally introduced into the syringe during sample loading should be removed at this phase. Firmly tapping on the side of the syringe will dislodge the bubbles from the syringe walls and float atop, allowing the user to push it out. A smaller needle (BD, 305122) and nylon tubing are used to transfer the sample from the syringe through the inlet and into the chamber. An important note during this process is to point the syringe upward to prevent the bubbles from getting trapped. Once a droplet of sample solutions forms on the end of the tubing, the tubing is ready to be connected with the appropriate inlet of the flow chamber. Forming droplets on both terminals (tubing ends and inlets) before connecting them is necessary as the liquid-liquid interface prevents air bubbles from entering the chamber. Since the syringes contain particles that will be loaded onto the same microfluidic pumps, their plumber level must be equalized. The flow assembly is then completed by loading the chamber onto the sample stage, securing the syringe onto the microfluidic pump and connecting the outlet with the waste reservoir using nylon tubing (Figure 38). Before initiating the experiment, a final thorough examination of the flow circuit should be carried out to ensure no potential issues, such as leakage, bubbles, chamber alignment, etc. 78 Figure 38. Microfluidic assembly. (a) Three buffer solutions are injected into the flow cell via a series of tubing and inlets. A single outlet combines all three flows, leading to the waste reservoir. (b) The contraption is then vertically installed onto the XYZ motorized stage. The red square indicates the area where the trap-forming objectives will be positioned during experimentation. 79 6 SINGLE-MOLECULE STRETCHING OF DNA As part of our system validation effort, we have replicated the classical DNA hairpin study conducted by Woodside et al.32. This widely adopted study serves as a benchmark for single-molecule optical tweezer systems, presenting a relatively straightforward system for construction and investigation. The DNA hairpin investigation shares numerous similarities with our current setup, including buffer system, experimental protocol, and chemical linkages. By reporting our replication data, we reinforce the accuracy and repeatability of the literature values, providing future investigations with a robust foundation upon which to build. Moreover, this replication study has shed light on weaknesses and potential issues within our current system, necessitating rectification before it can be fully functional for actual research purposes. 6.1 DNA force-extension measurement protocol Experimentation consists of two phases: the trapping phase and the experiment phase. During the trapping phase, an anti-dig particle is first trapped with the moving trap downstream from the flow. Then, the streptavidin-DNA particle is targeted by the fixed trap upstream. This trapping order reduces the chance of trapping multiple particles. Subsequently, the two trapped particles are translocated upstream of the middle channel, away from the junctions crowded with particles from the top and bottom flows (refer back to Figure 35). After adjusting the trapping depth to the middle of the surface-to-surface distance, the experimentation phase can begin, Stiffness calibration for the pair of particles is then carried out simultaneously at a 100 kHz sampling rate for 30 seconds. The output file is labelled with the âCALIBâ suffix, which will be recognized by our analysis program automatically. The moving trap is assigned a triangle waveform to periodically bring the anti-dig particle to proximity with the other DNA coated particle in the fixed trap, âfishingâ for a DNA molecule bridging between the probe particles. Here, a balance between the distance permitting a single tether to form and the vicinity where the chance of getting multiple tethers should be discovered. Typically, a standard triangle waveform is assigned at 0.5Hz frequency, allowing the user to detect and react to interactions between the two particles (by monitoring the displacement of these particles). However, specialized DNA structures may require different âfishingâ waveforms to shorten the moving-80 away period or lengthen the time the probe particles spend in close vicinity. We typically determine this steering distance and range using the first pair of particles. This steering range is highly dependent on the structure of the DNA molecule: contour length, special features such as knots, holiday junctions, etc. This is the reference for the subsequent particle pairs for the rest of the experiment. While it is possible to observe and detect multi-tethering between particles by observing particle displacement patterns, an absolute way to determine single tethering is to break off the tether post-experiment and seek a single drop of the signal to zero voltage. This means that the probe has broken off the tether and fallen back to the trap's centre. On the other hand, multi-stage voltage drops indicate multiple tethering, so the collected data should be treated differently. We found that holding the tether under high tension by moving the traps apart promotes the break-off to happen more often, speeding up this single-tether validation process. With our current DNA coating density, the same particle pair can form up to 3 tethering events before needing to be replaced. 6.2 Elasticity of double-stranded DNA DNA hairpin is a classical, well-explored system of study in single-molecule biophysics. It reveals fundamental mechanical aspects of DNA duplex stability, such as base-stacking and nearest-neighbour interactions. Upon completing the probing procedure, the experimental data is imported into Matlab, where a comprehensive processing and analysis pipeline has been developed. (Figure 39) The pipeline consists of three stages: (1) data flagging where baseline region and experimental region are selected from raw data and data structure is initialized, (2) parameters determination contains all calibration such as stiffness and optical sensitivity as well as data offset process, (3) data analysis utilizes parameters from previous stage to construct force-extension curve (FEC), detects rupture points for any dynamic structure (such as DNA hairpin) and performing worm-like chain (WLC) fitting. 81 Figure 39. Data processing pipeline. Raw data is imported into Matlab and is handled by a custom script with four main functions (import, flag calibrate and analyze). The raw voltage signal shows both particles being displaced from the trap centre as they move further apart. The portion of the voltage signal at which the tether has yet to form can then be used for baseline correction (Figure 40a, dark blue region). The formation of the tether is evident as the displacement signal started to change drastically during the âfishingâ cycle. (Figure 40a, light blue region) We also observe single breakage verifying single-molecule interaction. The accompanying calibration data is then assessed. The detector signal is sampled and filtered to fulfil the Nyquist criterion and avoid anti-aliasing effects. The stiffness calibration also reveals potential problems with the trapped particles, such as misshapes or more than one particle within the trap. It also highlights the resonance frequency of additional mechanical noises, which may impact data quality. Additionally, the baseline for experimental data is corrected to account for the interference signal coming from the polarization leakage between traps. 82 Figure 40. Typical data preprocessing display. (a) Data Flagging: user-pick OFFSET (dark blue) and EXPERIMENT (light blue) regions for subsequent analysis. (b) Stiffness Calibration: physical parameter calculation and qualitative analysis of systemic noise and other irregularities. The user can then decide to reject or accept the data set. Though the power spectrum for BY is not up to standard, this dataset is accepted since the deciding factor is power spectra in the X direction (AX and BX). Next, experimental data segments are broken down based on the steering cycle, with one capturing the movement of the particles away from each other (retract) and the other consisting of the movement of the particles coming closer together (approach). The retract portion often contains an unfolding event of a DNA hairpin as the tether is mechanically stretched beyond its default conformational limit. Vice versa, as the tether relaxed in the approach sub-cycle, the molecule returns to the entropically favourable folded hairpin state. These unfolding/refolding events have a certain characteristic saw-tooth pattern: a noticeable drop in applied force signalling the complete opening of the DNA hairpin. The force-extension curve (FEC) is plotted for all detected events. (Figure 41) 83 Figure 41. Force-extension curve series. Extension response for T30 hairpin when pulled apart (red) and return to the original position (blue). The hairpin unfolded at â 11 pN. Due to multiple tethers, initial retraction shows irregular rupture behaviour, breaking off at 15 pN. Refolding event is uncommon because of the large 30 Thymine nucleotides hairpin loop, making it challenging to observe at any extension beyond 950 nm. Inset is the 3D plot with the Z axis representing time, showing the sequence of the stretch cycles with rupture events. The FEC can be separated into 2 segments: pre-rupture, where the target molecule is extended to its contour length (~930 nm), and the post-rupture segment begins after the hairpin is ruptured and fully stretched. (Figure 42a) Several rupture detection methods have been proposed and made available within the community. However, they are not universal and are highly dependent on use cases and instrument setup. We have adapted the high-order polynomial segmentation method119 and an additional abrupt change detection function in our workflow to achieve the most accurate detection. Along with this core cross-validation, additional features such as short-lived events filtering, minimum force threshold and proximity search features have been added to make the detection as highly adaptable as possible for future use cases. 84 Figure 42. Rupture detection and WLC fit. (a) automatic rupture detection using a custom script to cross-validate multiple detection methods to improve accuracy and reduce false positives. The curve is split into pre/post-rupture (orange and yellow portion, respectively) segments and fits separately. (b) Worm-like chain fit for each segment. Each split segment of the FEC curve fits with the WLC model individually. Once base pair length values (ð¿ð¿ðððð) are calculated, we construct the WLC fit along with the original curve to 85 assess the goodness of fit. (Figure 42b) While the pre-rupture segments fit consistently well, the post-ruptureâs fit is often mismatched by 7-10 nm. A possible explanation is the unaccounted force calculation in the Y direction. The rejection of this force calculation stems from the fact that stiffness calibration, as seen in Figure 40b, reveals the existence of external noise in Y-directions. Thus, they cannot yet be included in the analysis. If a trapped pair of particles is not completely levelled, the force contribution consists of not only horizontal force but also vertical force. We expect the pre-rupture fit to improve once the noise source is identified and eliminated. To temporarily counter this problem, the trapped particles are carefully levelled to ensure minimal vertical force contribution. Figure 43. Histogram plot of fitted dsDNA base-pair length. The average ð³ð³ðððð (red line) is calculated to be 0.331 nm, where the number of accounted T30 hairpin rupture events n = 46 across 3 datasets of different DNA molecules. In the literature, the length of the DNA base pair has been measured using various techniques aside from an optical tweezer. X-ray crystallography provides a crystalline environment that minimizes the effects of hydration and ionic conditions. This results in a measurement of 0.34 nm with an error range of 0.01 nm/bp.120,121 Several AFM studies report similar numbers for the base pair length, slightly shorter at 0.32 nm/bp, and the error range, slightly higher at 0.02 nm/bp. The difference is attributed to temperature, sample preparation and technical challenges ranging from mechanical drift to image analysis.122â124 86 An optical tweezer has also been employed to verify this parameter. With measured helical rises ranging from 0.31 nm/bp to 0.34 nm/bp. The variability in these measurements largely stems from differences in ionic conditions and the loading forces applied by different experimental setups.125 In our study, the calculated base pair lengths distribution falls well within the range reported in the literature from X-ray crystallography, AFM, and other optical tweezer setups, all of which report base pair lengths near 0.34 nm/bp. This consistency indicates the successful execution of each phase in constructing and optimizing the optical tweezer instrument, including the precise alignment of optical components, effective noise management, and robust experimental design and data analysis. Consequently, the instrument has been validated and is deemed fully capable of investigating a wide range of biological systems with high precision. 87 7 DISCUSSION In pursuit of novel innovations within the realm of designer DNA probes and the investigation of under-explored aspects of protein dynamics and interactions, we have successfully developed a variation of the optical tweezer conceptânamely, a dual-trap optical tweezer system. This instrument is capable of high-resolution force measurements and allows for the precise manipulation of microscopic objects with a high degree of freedom. As a custom-built instrument, it offers complete control over software and hardware, facilitating rapid evaluation and integration of future upgrades or additional functionalities as experimental needs evolve. One major fallback for the instrument is its inability to perform force clamp - the ability to maintain a tension force for an extended period accurately. This enables studying the kinetic process of the multi-state or multi-conformation molecules. Force clamp can be done by implementing a direct force feedback component where an additional controller and more advanced beam steering system are required.126 Passive force clamp is an alternative approach that does not require additional hardware upgrades. This is done by utilizing the anharmonic region of the trap â the region where the force applied on the trapped particle near its highest before it falls out of the trap. As long as the particle is in this region, its displacement (reported by the photo sensor) is temporarily decoupled with the force applied. The particle is essentially force-clamped. Adjusting the constant force can be done by simply varying the laser intensity. 104 The implementation requires extensive assessment of trapping potential at different laser powers to explore the non-linear region. This ultimately lies beyond the scope of this thesis and, therefore, has not been implemented at the current time. Since the force clamp is needed for the comparison of ð¹ð¹1/2 value analyzed from the validation T30 hairpin, we have moved away from using this value and pivoted to fitting persistent length and contour length parameters from the force-extension data. As the mentioned parameters is well matched to the literature, this serves as a solid alternative to validate our instrument. There are a few important recommendations to improve experiment output and quality. These should also be suggestions to improve the instrumentâs capabilities further. Firstly, position sensing detectors should be considered over quadrant photodiode for a more accurate force measurement105. This is due to the sensitivity of QPD to particle size and overall interferometric pattern. It fails to present the centroid of the light trap. This will result in more 88 dispersed positional data and incorrect force measurement. On the other hand, PSDs are more versatile in dealing with particle shapes and sizes. Since it senses over a continuous area, it provides a more accurate representation of trap centroid regardless of size and shape. Secondly, additional noise assessment can be carried out to improve force-resolving capabilities further. Laser characterization is needed to determine the impact of frequency noise on trapping power. Frequency noise (or phase noise) results from imperfect excitation of a target frequency. Variations of frequency and phase may result in laser power fluctuations, leading to an unstable trap and producing unwanted random motions by trapped particles. Additionally, phase difference causes destructive and constructive interferences that alter the signal detected by photodiodes, disrupting temporal coherence and decreasing the signal-to-noise ratio. This negatively impacts power spectral density analysis, leading to mis-calibration of the trapped particles. Another noise source comes from mechanical vibrations in the Y-axis, as seen in the power spectrum AY and BY in Figure 41 from Chapter 6. While the contribution of the Y-force component to the overall force curve is generally minimal, it varies significantly daily. The uncertainty of this noiseâs impact on the experiment makes it the top priority for our subsequent upgrade effort. We suspected that the multi-axis stage mounting mechanism, specifically the stage arm, did not provide enough rigidity and was prone to vertical (Y-axis) vibration. A new mounting solution is currently being worked on to remedy this issue. Thirdly, much attention should be paid to particle preparation and microfluidic management. One may face a significant setback in time and effort if the particles are under/over-coated and have irregular fluidic flow. Typically, having a low density of DNA on a particle surface may reduce the chance of multi-tether formation. We find that the tethering time significantly increases, diminishing the amount of data output. Conversely, a densely coated surface encourages multiple spontaneous tether formations. It could pose a problem to the validity of the collected data, and, more importantly, it could be challenging for the user to detect. A balanced coating will typically produce at least 1 interaction per minute. This allows users to collect hundreds of interactions per experiment in order. Coating optimization is highly dependent on the target structure. We found that 5-10ng of DNA per 10 μL of stock 0.1% particle solution (1 μm diameter) is a good starting point. 89 Additionally, the user should verify the tether after every raw measurement. This can be done by stretching the target molecule and observing the tether breakage event for a single drop in positional signal as the particles break away from the tether and return to the trapping centre. This signifies a single tether formation between the particles. The optical tweezer system has already become an indispensable tool in our laboratory. It allows us to validate experimental results from other single-molecule techniques, such as single-molecule fluorescence and atomic force microscopy. Given our confidence in the system's capabilities, we have studied the artificial reproduction of fundamental intermolecular forces, such as catch bonds. Catch bonds are noncovalent bonds that exhibit an increased lifetime when a force is applied. It exists in various biological interactions ranging from leukocyte rolling adhesion behaviour as they traverse the blood vessel to E. coli adhesion on the human intestinal wall. A secondary structure of DNA can be designed to mimic this behaviour. Further fine-tuning parameters such as DNA sequences and base pair length alters the force threshold of the catch bond interaction. This enables a faithful and systematic study of all known variations of catch bonds with minimal effort. Lastly, the newly constructed optical tweezer system allowed us to delve into intriguing sequence-based DNA behaviours like twisting and knotting. Psuedo-knot is a regulator and promoter in viral RNA translation and replication.77 Single-molecule analysis using optical tweezers provides deeper insights into the biomechanical properties of these structures. This enhanced understanding can inform the development of gene therapies, where custom-designed DNA sequences could specifically target and disrupt these critical structures. 90 8 CONCLUSION In conclusion, I successfully built a dual-trap optical tweezer system capable of high-resolution force measurements and exceptional adaptability to advance the development of novel designer DNA probes. A comprehensive standard operating protocol has been established, covering all necessary preparation procedures for DNA characterization experiments. The instrument has undergone rigorous calibration and validation by replicating established protocols, ensuring its performance meets published standards. Unlike commercial systems, our setup offers comparable capabilities with more significant data collection and post-processing flexibility, granting users full autonomy. Additionally, the system can be easily modified or upgraded, integrating fluorescence capabilities, advanced steering techniques (AOD, SLM), or feedback systems (temperature, drift) without the limitations typically imposed by commercial alternatives. 91 REFERENCES (1) Ashkin, A.; Dziedzic, J. M.; Bjorkholm, J. E.; Chu, S. Observation of a Single-Beam Gradient Force Optical Trap for Dielectric Particles. 3. (2) Kepler, J. De Cometis Libelli Tres; 1619. (3) Maxwell, J. C. A Treatise on Electricity and Magnetism; Clarendon Press, 1873. (4) Nichols, E. F.; Hull, G. F. A Preliminary Communication on the Pressure of Heat and Light Radiation. Phys. Rev. Ser. I 1901, 13 (5), 307â320. https://doi.org/10.1103/PhysRevSeriesI.13.307. (5) Nieminen, T. A.; Stilgoe, A. B.; Heckenberg, N. R.; Rubinsztein-Dunlop, H. H. Approximate and Exact Modeling of Optical Trapping; Dholakia, K., Spalding, G. C., Eds.; San Diego, California, 2010; p 77622V. https://doi.org/10.1117/12.861880. (6) Lock, J. A.; Gouesbet, G. Generalized LorenzâMie Theory and Applications. J. Quant. Spectrosc. Radiat. Transf. 2009, 110 (11), 800â807. https://doi.org/10.1016/j.jqsrt.2008.11.013. (7) Gouesbet, G.; Gréhan, G. Generalized Lorenz-Mie Theories; Springer Berlin Heidelberg: Berlin, Heidelberg, 2011. https://doi.org/10.1007/978-3-642-17194-9. (8) Mazolli, A.; Maia Neto, P. A.; Nussenzveig, H. M. Theory of Trapping Forces in Optical Tweezers. Proc. R. Soc. Lond. Ser. Math. Phys. Eng. Sci. 2003, 459 (2040), 3021â3041. https://doi.org/10.1098/rspa.2003.1164. (9) Nieminen, T. A.; Loke, V. L. Y.; Stilgoe, A. B.; Knöner, G.; BraÅczyk, A. M.; Heckenberg, N. R.; Rubinsztein-Dunlop, H. Optical Tweezers Computational Toolbox. J. Opt. Pure Appl. Opt. 2007, 9 (8), S196. https://doi.org/10.1088/1464-4258/9/8/S12. (10) Waterman, P. C. Matrix Formulation of Electromagnetic Scattering. Proc. IEEE 1965, 53 (8), 805â812. https://doi.org/10.1109/PROC.1965.4058. (11) Bishop, A. I.; Nieminen, T. A.; Heckenberg, N. R.; Rubinsztein-Dunlop, H. Optical Application and Measurement of Torque on Microparticles of Isotropic Nonabsorbing 92 Material. Phys. Rev. A 2003, 68 (3), 033802. https://doi.org/10.1103/PhysRevA.68.033802. (12) Borghese, F.; Denti, P.; Saija, R.; Iatì, M. A. Optical Trapping of Nonspherical Particles in the T-Matrix Formalism. Opt. Express 2007, 15 (19), 11984â11998. https://doi.org/10.1364/OE.15.011984. (13) Nieminen, T.; Loke, V.; Stilgoe, A.; Heckenberg, N.; Rubinsztein-Dunlop, H. T-Matrix Method for Modelling Optical Tweezers. J. Mod. Opt. 2011, 58. https://doi.org/10.1080/09500340.2010.528565. (14) Pesce, G.; Jones, P. H.; Maragò, O. M.; Volpe, G. Optical Tweezers: Theory and Practice. Eur. Phys. J. Plus 2020, 135 (12), 949. https://doi.org/10.1140/epjp/s13360-020-00843-5. (15) Ashkin, A. Forces of a Single-Beam Gradient Laser Trap on a Dielectric Sphere in the Ray Optics Regime. Biophys. J. 1992, 61 (2), 569â582. https://doi.org/10.1016/S0006-3495(92)81860-X. (16) Ma, J.; Bai, L.; Wang, M. D. Transcription Under Torsion. Science 2013, 340 (6140), 1580â1583. https://doi.org/10.1126/science.1235441. (17) Shaevitz, J. W.; Abbondanzieri, E. A.; Landick, R.; Block, S. M. Backtracking by Single RNA Polymerase Molecules Observed at Near-Base-Pair Resolution. Nature 2003, 426 (6967), 684â687. https://doi.org/10.1038/nature02191. (18) van Mameren, J.; Gross, P.; Farge, G.; Hooijman, P.; Modesti, M.; Falkenberg, M.; Wuite, G. J. L.; Peterman, E. J. G. Unraveling the Structure of DNA during Overstretching by Using Multicolor, Single-Molecule Fluorescence Imaging. Proc. Natl. Acad. Sci. 2009, 106 (43), 18231â18236. https://doi.org/10.1073/pnas.0904322106. (19) Mameren, J. van; Modesti, M.; Kanaar, R.; Wyman, C.; Wuite, G. J. L.; Peterman, E. J. G. Dissecting Elastic Heterogeneity along DNA Molecules Coated Partly with Rad51 Using Concurrent Fluorescence Microscopy and Optical Tweezers. Biophys. J. 2006, 91 (8), L78âL80. https://doi.org/10.1529/biophysj.106.089466. 93 (20) Lee, J. Y.; Wang, F.; Fazio, T.; Wind, S.; Greene, E. C. Measuring Intermolecular Rupture Forces with a Combined TIRF-Optical Trap Microscope and DNA Curtains. Biochem. Biophys. Res. Commun. 2012, 426 (4), 565â570. https://doi.org/10.1016/j.bbrc.2012.08.127. (21) Lee, K. S.; Balci, H.; Jia, H.; Lohman, T. M.; Ha, T. Direct Imaging of Single UvrD Helicase Dynamics on Long Single-Stranded DNA. Nat. Commun. 2013, 4 (1), 1878. https://doi.org/10.1038/ncomms2882. (22) Comstock, M. J.; Ha, T.; Chemla, Y. R. Ultrahigh-Resolution Optical Trap with Single-Fluorophore Sensitivity. Nat. Methods 2011, 8 (4), 335â340. https://doi.org/10.1038/nmeth.1574. (23) Whitley, K. D.; Comstock, M. J.; Chemla, Y. R. High-Resolution âFleezersâ: Dual-Trap Optical Tweezers Combined with Single-Molecule Fluorescence Detection. In Optical Tweezers: Methods and Protocols; Gennerich, A., Ed.; Methods in Molecular Biology; Springer: New York, NY, 2017; pp 183â256. https://doi.org/10.1007/978-1-4939-6421-5_8. (24) Brennan, L. D.; Forties, R. A.; Patel, S. S.; Wang, M. D. DNA Looping Mediates Nucleosome Transfer. Nat. Commun. 2016, 7 (1), 13337. https://doi.org/10.1038/ncomms13337. (25) Forth, S.; Deufel, C.; Patel, S. S.; Wang, M. D. Direct Measurements of Torque during Holliday Junction Migration. Biophys. J. 2011, 101 (2), L5-7. https://doi.org/10.1016/j.bpj.2011.05.066. (26) Wang, M. D.; Yin, H.; Landick, R.; Gelles, J.; Block, S. M. Stretching DNA with Optical Tweezers. Biophys. J. 1997, 72 (3), 1335â1346. https://doi.org/10.1016/S0006-3495(97)78780-0. (27) F. Bazoni, R.; M. Lima, C. H.; B. Ramos, E.; S. Rocha, M. Force-Dependent Persistence Length of DNAâIntercalator Complexes Measured in Single Molecule Stretching Experiments. Soft Matter 2015, 11 (21), 4306â4314. https://doi.org/10.1039/C5SM00706B. 94 (28) McCauley, M. J.; Williams, M. C. Mechanisms of DNA Binding Determined in Optical Tweezers Experiments. Biopolymers 2007, 85 (2), 154â168. https://doi.org/10.1002/bip.20622. (29) Wenner, J. R.; Williams, M. C.; Rouzina, I.; Bloomfield, V. A. Salt Dependence of the Elasticity and Overstretching Transition of Single DNA Molecules. Biophys. J. 2002, 82 (6), 3160â3169. https://doi.org/10.1016/S0006-3495(02)75658-0. (30) Seol, Y.; Skinner, G. M.; Visscher, K.; Buhot, A.; Halperin, A. Stretching of Homopolymeric RNA Reveals Single-Stranded Helices and Base-Stacking. Phys. Rev. Lett. 2007, 98 (15), 158103. https://doi.org/10.1103/PhysRevLett.98.158103. (31) Kilchherr, F.; Wachauf, C.; Pelz, B.; Rief, M.; Zacharias, M.; Dietz, H. Single-Molecule Dissection of Stacking Forces in DNA. Science 2016, 353 (6304), aaf5508. https://doi.org/10.1126/science.aaf5508. (32) Woodside, M. T.; Behnke-Parks, W. M.; Larizadeh, K.; Travers, K.; Herschlag, D.; Block, S. M. Nanomechanical Measurements of the Sequence-Dependent Folding Landscapes of Single Nucleic Acid Hairpins. Proc. Natl. Acad. Sci. U. S. A. 2006, 103 (16), 6190â6195. https://doi.org/10.1073/pnas.0511048103. (33) Green, L.; Kim, C.-H.; Bustamante, C.; Tinoco, I. Characterization of the Mechanical Unfolding of RNA Pseudoknots. J. Mol. Biol. 2008, 375 (2), 511â528. https://doi.org/10.1016/j.jmb.2007.05.058. (34) McCauley, M. J.; Williams, M. C. Optical Tweezers Experiments Resolve Distinct Modes of DNA-Protein Binding. Biopolymers 2009, 91 (4), 265â282. https://doi.org/10.1002/bip.21123. (35) Mohapatra, S.; Lin, C.-T.; Feng, X. A.; Basu, A.; Ha, T. Single-Molecule Analysis and Engineering of DNA Motors. Chem. Rev. 2020, 120 (1), 36â78. https://doi.org/10.1021/acs.chemrev.9b00361. (36) Castelino, K.; Satyanarayana, S.; Sitti, M. Manufacturing of Two and Three-Dimensional Micro/Nanostructures by Integrating Optical Tweezers with Chemical 95 Assembly. Robotica 2005, 23 (4), 435â439. https://doi.org/10.1017/S0263574704000864. (37) Sinclair, G.; Jordan, P.; Courtial, J.; Padgett, M.; Cooper, J.; Laczik, Z. J. Assembly of 3-Dimensional Structures Using Programmable Holographic Optical Tweezers. Opt. Express 2004, 12 (22), 5475â5480. https://doi.org/10.1364/OPEX.12.005475. (38) Koirala, D.; Shrestha, P.; Emura, T.; Hidaka, K.; Mandal, S.; Endo, M.; Sugiyama, H.; Mao, H. Single-Molecule Mechanochemical Sensing Using DNA Origami Nanostructures. Angew. Chem. Int. Ed. 2014, 53 (31), 8137â8141. https://doi.org/10.1002/anie.201404043. (39) Choquet, D.; Felsenfeld, D. P.; Sheetz, M. P. Extracellular Matrix Rigidity Causes Strengthening of IntegrinâCytoskeleton Linkages. Cell 1997, 88 (1), 39â48. https://doi.org/10.1016/S0092-8674(00)81856-5. (40) Svoboda, K.; Schmidt, C. F.; Schnapp, B. J.; Block, S. M. Direct Observation of Kinesin Stepping by Optical Trapping Interferometry. Nature 1993, 365 (6448), 721â727. https://doi.org/10.1038/365721a0. (41) Kuo, S. C.; Sheetz, M. P. Force of Single Kinesin Molecules Measured with Optical Tweezers. Science 1993, 260 (5105), 232â234. https://doi.org/10.1126/science.8469975. (42) Mickolajczyk, K. J.; Shelton, P. M. M.; Grasso, M.; Cao, X.; Warrington, S. E.; Aher, A.; Liu, S.; Kapoor, T. M. Force-Dependent Stimulation of RNA Unwinding by SARS-CoV-2 Nsp13 Helicase. Biophys. J. 2021, 120 (6), 1020â1030. https://doi.org/10.1016/j.bpj.2020.11.2276. (43) Brouwer, I.; Moschetti, T.; Candelli, A.; Garcin, E. B.; Modesti, M.; Pellegrini, L.; Wuite, G. J.; Peterman, E. J. Two Distinct Conformational States Define the Interaction of Human RAD51-ATP with Single-Stranded DNA. EMBO J. 2018, 37 (7), e98162. https://doi.org/10.15252/embj.201798162. (44) Stigler, J.; Ziegler, F.; Gieseke, A.; Gebhardt, J. C. M.; Rief, M. The Complex Folding Network of Single Calmodulin Molecules. Science 2011, 334 (6055), 512â516. https://doi.org/10.1126/science.1207598. 96 (45) Direct observation of multiple misfolding pathways in a single prion protein molecule. https://doi.org/10.1073/pnas.1107736109. (46) Neupane, K.; Woodside, M. T. Hidden Depths of Protein Folding. Nat. Phys. 2023, 19 (1), 8â9. https://doi.org/10.1038/s41567-022-01828-0. (47) Curtis, J. E.; Koss, B. A.; Grier, D. G. Dynamic Holographic Optical Tweezers. Opt. Commun. 2002, 207 (1), 169â175. https://doi.org/10.1016/S0030-4018(02)01524-9. (48) Visscher, K.; Gross, S. P.; Block, S. M. Construction of Multiple-Beam Optical Traps with Nanometer-Resolution Position Sensing. IEEE J. Sel. Top. Quantum Electron. 1996, 2 (4), 1066â1076. https://doi.org/10.1109/2944.577338. (49) Chapin, S. C.; Germain, V.; Dufresne, E. R. Automated Trapping, Assembly, and Sorting with Holographic Optical Tweezers. Opt. Express 2006, 14 (26), 13095â13100. https://doi.org/10.1364/OE.14.013095. (50) Li, C.-Y.; Cao, D.; Qi, C.-B.; Kang, Y.-F.; Song, C.-Y.; Xu, D.-D.; Zheng, B.; Pang, D.-W.; Tang, H.-W. Combining Holographic Optical Tweezers with Upconversion Luminescence Encoding: Imaging-Based Stable Suspension Array for Sensitive Responding of Dual Cancer Biomarkers. Anal. Chem. 2018, 90 (4), 2639â2647. https://doi.org/10.1021/acs.analchem.7b04299. (51) Paterson, L.; MacDonald, M. P.; Arlt, J.; Sibbett, W.; Bryant, P. E.; Dholakia, K. Controlled Rotation of Optically Trapped Microscopic Particles. Science 2001, 292 (5518), 912â914. https://doi.org/10.1126/science.1058591. (52) Hosseinzadeh, M.; Hajizadeh, F.; Habibi, M.; Milani Moghaddam, H.; S. Reihani, S. N. Optimized Rotation of an Optically Trapped Particle for Micro Mixing. Appl. Phys. Lett. 2018, 113 (22), 223701. https://doi.org/10.1063/1.5053971. (53) Terray, A.; Oakey, J.; Marr, D. Fabrication of Linear Colloidal Structures for Microfluidic Applications. Appl. Phys. Lett. 2002, 81, 1555â1557. https://doi.org/10.1063/1.1503176. 97 (54) Zhang, Y.; Min, C.; Dou, X.; Wang, X.; Urbach, H. P.; Somekh, M. G.; Yuan, X. Plasmonic Tweezers: For Nanoscale Optical Trapping and Beyond. Light Sci. Appl. 2021, 10 (1), 59. https://doi.org/10.1038/s41377-021-00474-0. (55) Li, H.; Ren, Y.; Li, Y.; He, M.; Gao, B.; Qi, H. Nanoparticle Manipulation Using Plasmonic Optical Tweezers Based on Particle Sizes and Refractive Indices. Opt. Express 2022, 30 (19), 34092. https://doi.org/10.1364/OE.468024. (56) Kotsifaki, D. G.; Chormaic, S. N. Plasmonic Optical Tweezers Based on Nanostructures: Fundamentals, Advances and Prospects. Nanophotonics 2019, 8 (7), 1227â1245. https://doi.org/10.1515/nanoph-2019-0151. (57) Yoshikawa, T.; Tamura, M.; Tokonami, S.; Iida, T. Optical Trap-Mediated High-Sensitivity Nanohole Array Biosensors with Random Nanospikes. J. Phys. Chem. Lett. 2017, 8 (2), 370â374. https://doi.org/10.1021/acs.jpclett.6b02262. (58) Belkin, M.; Chao, S.-H.; Jonsson, M. P.; Dekker, C.; Aksimentiev, A. Plasmonic Nanopores for Trapping, Controlling Displacement, and Sequencing of DNA. ACS Nano 2015, 9 (11), 10598â10611. https://doi.org/10.1021/acsnano.5b04173. (59) Wells, R. D. Non-B DNA Conformations, Mutagenesis and Disease. Trends Biochem. Sci. 2007, 32 (6), 271â278. https://doi.org/10.1016/j.tibs.2007.04.003. (60) Zhao, J.; Bacolla, A.; Wang, G.; Vasquez, K. M. Non-B DNA Structure-Induced Genetic Instability and Evolution. Cell. Mol. Life Sci. CMLS 2009, 67 (1), 43â62. https://doi.org/10.1007/s00018-009-0131-2. (61) Marquis Gacy, A.; Goellner, G.; JuraniÄ, N.; Macura, S.; McMurray, C. T. Trinucleotide Repeats That Expand in Human Disease Form Hairpin Structures in Vitro. Cell 1995, 81 (4), 533â540. https://doi.org/10.1016/0092-8674(95)90074-8. (62) Kato, T.; Franconi, C. P.; Sheridan, M. B.; Hacker, A. M.; Inagakai, H.; Glover, T. W.; Arlt, M. F.; Drabkin, H. A.; Gemmill, R. M.; Kurahashi, H.; Emanuel, B. S. Analysis of the t(3;8) of Hereditary Renal Cell Carcinoma: A Palindrome-Mediated Translocation. Cancer Genet. 2014, 207 (4), 133â140. https://doi.org/10.1016/j.cancergen.2014.03.004. 98 (63) Dhanasekaran, R.; Deutzmann, A.; Mahauad-Fernandez, W. D.; Hansen, A. S.; Gouw, A. M.; Felsher, D. W. The MYC Oncogene â the Grand Orchestrator of Cancer Growth and Immune Evasion. Nat. Rev. Clin. Oncol. 2022, 19 (1), 23â36. https://doi.org/10.1038/s41571-021-00549-2. (64) Melaiu, O.; Mina, M.; Chierici, M.; Boldrini, R.; Jurman, G.; Romania, P.; DâAlicandro, V.; Benedetti, M. C.; Castellano, A.; Liu, T.; Furlanello, C.; Locatelli, F.; Fruci, D. PD-L1 Is a Therapeutic Target of the Bromodomain Inhibitor JQ1 and, Combined with HLA Class I, a Promising Prognostic Biomarker in Neuroblastoma. Clin. Cancer Res. 2017, 23 (15), 4462â4472. https://doi.org/10.1158/1078-0432.CCR-16-2601. (65) DeJesus-Hernandez, M.; Mackenzie, I. R.; Boeve, B. F.; Boxer, A. L.; Baker, M.; Rutherford, N. J.; Nicholson, A. M.; Finch, N. A.; Gilmer, H. F.; Adamson, J.; Kouri, N.; Wojtas, A.; Sengdy, P.; Hsiung, G.-Y. R.; Karydas, A.; Seeley, W. W.; Josephs, K. A.; Coppola, G.; Geschwind, D. H.; Wszolek, Z. K.; Feldman, H.; Knopman, D.; Petersen, R.; Miller, B. L.; Dickson, D.; Boylan, K.; Graff-Radford, N.; Rademakers, R. Expanded GGGGCC Hexanucleotide Repeat in Non-Coding Region of C9ORF72 Causes Chromosome 9p-Linked Frontotemporal Dementia and Amyotrophic Lateral Sclerosis. Neuron 2011, 72 (2), 245â256. https://doi.org/10.1016/j.neuron.2011.09.011. (66) Reddy, K.; Zamiri, B.; Stanley, S. Y. R.; Macgregor, R. B.; Pearson, C. E. The Disease-Associated r(GGGGCC)n Repeat from the C9orf72 Gene Forms Tract Length-Dependent Uni- and Multimolecular RNA G-Quadruplex Structures. J. Biol. Chem. 2013, 288 (14), 9860â9866. https://doi.org/10.1074/jbc.C113.452532. (67) Piazza, A.; Boulé, J.-B.; Lopes, J.; Mingo, K.; Largy, E.; Teulade-Fichou, M.-P.; Nicolas, A. Genetic Instability Triggered by G-Quadruplex Interacting Phen-DC Compounds in Saccharomyces Cerevisiae. Nucleic Acids Res. 2010, 38 (13), 4337â4348. https://doi.org/10.1093/nar/gkq136. (68) Smith, J. S.; Chen, Q.; Yatsunyk, L. A.; Nicoludis, J. M.; Garcia, M. S.; Kranaster, R.; Balasubramanian, S.; Monchaud, D.; Teulade-Fichou, M.-P.; Abramowitz, L.; Schultz, D. C.; Johnson, F. B. Rudimentary G-QuadruplexâBased Telomere Capping in 99 Saccharomyces Cerevisiae. Nat. Struct. Mol. Biol. 2011, 18 (4), 478â485. https://doi.org/10.1038/nsmb.2033. (69) Maizels, N. Genomic Stability: FANCJ-Dependent G4 DNA Repair. Curr. Biol. CB 2008, 18 (14), R613âR614. https://doi.org/10.1016/j.cub.2008.06.011. (70) Wu, Y.; Brosh, R. M. G-Quadruplex Nucleic Acids and Human Disease. FEBS J. 2010, 277 (17), 3470â3488. https://doi.org/10.1111/j.1742-4658.2010.07760.x. (71) Lopes, J.; Piazza, A.; Bermejo, R.; Kriegsman, B.; Colosio, A.; Teulade-Fichou, M.-P.; Foiani, M.; Nicolas, A. G-Quadruplex-Induced Instability during Leading-Strand Replication. EMBO J. 2011, 30 (19), 4033â4046. https://doi.org/10.1038/emboj.2011.316. (72) Chen, M. C.; Tippana, R.; Demeshkina, N. A.; Murat, P.; Balasubramanian, S.; Myong, S.; Ferré-DâAmaré, A. R. Structural Basis of G-Quadruplex Unfolding by the DEAH/RHA Helicase DHX36. Nature 2018, 558 (7710), 465â469. https://doi.org/10.1038/s41586-018-0209-9. (73) Wang, Y.-R.; Guo, T.-T.; Zheng, Y.-T.; Lai, C.-W.; Sun, B.; Xi, X.-G.; Hou, X.-M. Replication Protein A Plays Multifaceted Roles Complementary to Specialized Helicases in Processing G-Quadruplex DNA. iScience 2021, 24 (5), 102493. https://doi.org/10.1016/j.isci.2021.102493. (74) Burge, S.; Parkinson, G. N.; Hazel, P.; Todd, A. K.; Neidle, S. Quadruplex DNA: Sequence, Topology and Structure. Nucleic Acids Res. 2006, 34 (19), 5402â5415. https://doi.org/10.1093/nar/gkl655. (75) Cheng, Y.; Zhang, Y.; You, H. Characterization of G-Quadruplexes Folding/Unfolding Dynamics and Interactions with Proteins from Single-Molecule Force Spectroscopy. Biomolecules 2021, 11 (11), 1579. https://doi.org/10.3390/biom11111579. (76) Barry, J. K.; Miller, W. A. A â1 Ribosomal Frameshift Element That Requires Base Pairing across Four Kilobases Suggests a Mechanism of Regulating Ribosome and Replicase Traffic on a Viral RNA. Proc. Natl. Acad. Sci. 2002, 99 (17), 11133â11138. https://doi.org/10.1073/pnas.162223099. 100 (77) Brierley, I.; Pennell, S.; Gilbert, R. J. C. Viral RNA Pseudoknots: Versatile Motifs in Gene Expression and Replication. Nat. Rev. Microbiol. 2007, 5 (8), 598â610. https://doi.org/10.1038/nrmicro1704. (78) Vicens, Q.; Cech, T. R. Atomic Level Architecture of Group I Introns Revealed. Trends Biochem. Sci. 2006, 31 (1), 41â51. https://doi.org/10.1016/j.tibs.2005.11.008. (79) Tanner, D. R.; Dewey, J. D.; Miller, M. R.; Buskirk, A. R. Genetic Analysis of the Structure and Function of Transfer Messenger RNA Pseudoknot 1 *. J. Biol. Chem. 2006, 281 (15), 10561â10566. https://doi.org/10.1074/jbc.M600167200. (80) Dinman, J. D. Mechanisms and Implications of Programmed Translational Frameshifting. Wiley Interdiscip. Rev. RNA 2012, 3 (5), 661â673. https://doi.org/10.1002/wrna.1126. (81) Poltronieri, P.; Sun, B.; Mallardo, M. RNA Viruses: RNA Roles in Pathogenesis, Coreplication and Viral Load. Curr. Genomics 2015, 16 (5), 327â335. https://doi.org/10.2174/1389202916666150707160613. (82) Omar, S. I.; Zhao, M.; Sekar, R. V.; Moghadam, S. A.; Tuszynski, J. A.; Woodside, M. T. Modeling the Structure of the Frameshift-Stimulatory Pseudoknot in SARS-CoV-2 Reveals Multiple Possible Conformers. PLoS Comput. Biol. 2021, 17 (1), e1008603. https://doi.org/10.1371/journal.pcbi.1008603. (83) Neupane, K.; Zhao, M.; Lyons, A.; Munshi, S.; Ileperuma, S. M.; Ritchie, D. B.; Hoffer, N. Q.; Narayan, A.; Woodside, M. T. Structural Dynamics of Single SARS-CoV-2 Pseudoknot Molecules Reveal Topologically Distinct Conformers. Nat. Commun. 2021, 12 (1), 4749. https://doi.org/10.1038/s41467-021-25085-6. (84) Kelly, J. A.; Woodside, M. T.; Dinman, J. D. Programmed â1 Ribosomal Frameshifting in Coronaviruses: A Therapeutic Target. Virology 2021, 554, 75â82. https://doi.org/10.1016/j.virol.2020.12.010. (85) Halma, M. T. J.; Ritchie, D. B.; Woodside, M. T. Conformational Shannon Entropy of mRNA Structures from Force Spectroscopy Measurements Predicts the Efficiency of 101 $ensuremath{-}1$ Programmed Ribosomal Frameshift Stimulation. Phys. Rev. Lett. 2021, 126 (3), 038102. https://doi.org/10.1103/PhysRevLett.126.038102. (86) De Vlaminck, I.; Henighan, T.; van Loenhout, M. T. J.; Pfeiffer, I.; Huijts, J.; Kerssemakers, J. W. J.; Katan, A. J.; van Langen-Suurling, A.; van der Drift, E.; Wyman, C.; Dekker, C. Highly Parallel Magnetic Tweezers by Targeted DNA Tethering. Nano Lett. 2011, 11 (12), 5489â5493. https://doi.org/10.1021/nl203299e. (87) Sitters, G.; Kamsma, D.; Thalhammer, G.; Ritsch-Marte, M.; Peterman, E. J. G.; Wuite, G. J. L. Acoustic Force Spectroscopy. Nat. Methods 2015, 12 (1), 47â50. https://doi.org/10.1038/nmeth.3183. (88) Cnossen, J. P.; Dulin, D.; Dekker, N. H. An Optimized Software Framework for Real-Time, High-Throughput Tracking of Spherical Beads. Rev. Sci. Instrum. 2014, 85 (10), 103712. https://doi.org/10.1063/1.4898178. (89) Català , F.; Marsà , F.; Montes-Usategui, M.; Farré, A.; Martín-Badosa, E. Influence of Experimental Parameters on the Laser Heating of an Optical Trap. Sci. Rep. 2017, 7 (1), 16052. https://doi.org/10.1038/s41598-017-15904-6. (90) Liu, Y.; Cheng, D. K.; Sonek, G. J.; Berns, M. W.; Chapman, C. F.; Tromberg, B. J. Evidence for Localized Cell Heating Induced by Infrared Optical Tweezers. Biophys. J. 1995, 68 (5), 2137â2144. https://doi.org/10.1016/S0006-3495(95)80396-6. (91) Blázquez-Castro, A. Optical Tweezers: Phototoxicity and Thermal Stress in Cells and Biomolecules. Micromachines 2019, 10 (8), 507. https://doi.org/10.3390/mi10080507. (92) Neuman, K. C.; Chadd, E. H.; Liou, G. F.; Bergman, K.; Block, S. M. Characterization of Photodamage to Escherichia Coli in Optical Traps. Biophys. J. 1999, 77 (5), 2856â2863. https://doi.org/10.1016/S0006-3495(99)77117-1. (93) Lahenkari, P. P. Adapting Atomic Force Microscopy for Cell Biology. https://doi.org/10.1016/S0304-3991(99)00138-2. (94) Thie, M.; Rospel, R.; Dettmann, W.; Benoit, M.; Ludwig, M.; Gaub, H. E.; Denker, H. W. Interactions between Trophoblast and Uterine Epithelium: Monitoring of Adhesive 102 Forces. Hum. Reprod. 1998, 13 (11), 3211â3219. https://doi.org/10.1093/humrep/13.11.3211. (95) Neuman, K. C.; Nagy, A. Single-Molecule Force Spectroscopy: Optical Tweezers, Magnetic Tweezers and Atomic Force Microscopy. Nat. Methods 2008, 5 (6), 491â505. https://doi.org/10.1038/nmeth.1218. (96) Bustamante, C.; Chemla, Y. R.; Moffitt, J. R. High-Resolution Dual-Trap Optical Tweezers with Differential Detection: Instrument Design. Cold Spring Harb. Protoc. 2009, 2009 (10), pdb.ip73. https://doi.org/10.1101/pdb.ip73. (97) Soni, G. V.; Hameed, F. M.; Roopa, T.; Shivashankar, G. V. Development of an Optical Tweezer Combined with Micromanipulation for DNA and Protein Nanobioscience. Curr. Sci. 2002, 83 (12), 7. (98) Enger, J.; Goksör, M.; Ramser, K.; Hagberg, P.; Hanstorp, D. Optical Tweezers Applied to a Microfluidic System. Lab Chip 2004, 4 (3), 196â200. https://doi.org/10.1039/B307960K. (99) Shane, J. C.; Mazilu, M.; Lee, W. M.; Dholakia, K. Effect of Pulse Temporal Shape on Optical Trapping and Impulse Transfer Using Ultrashort Pulsed Lasers. Opt. Express 2010, 18 (7), 7554â7568. https://doi.org/10.1364/OE.18.007554. (100) Goswami, D. Understanding Femtosecond Optical Tweezers: The Critical Role of Nonlinear Interactions. J. Phys. Conf. Ser. 2021, 1919 (1), 012013. https://doi.org/10.1088/1742-6596/1919/1/012013. (101) Theofanidou, E.; Laurence, W.; Hossack, W. J.; Arlt, J. Spherical Aberration Correction for Optical Tweezers. https://doi.org/10.1016/j.optcom.2004.03.009. (102) Viana, N. B.; Rocha, M. S.; Mesquita, O. N.; Mazolli, A.; Maia Neto, P. A.; Nussenzveig, H. M. Towards Absolute Calibration of Optical Tweezers. Phys. Rev. E 2007, 75 (2), 021914. https://doi.org/10.1103/PhysRevE.75.021914. (103) Yehoshua, S.; Pollari, R.; Milstein, J. N. Axial Optical Traps: A New Direction for Optical Tweezers. Biophys. J. 2015, 108 (12), 2759â2766. https://doi.org/10.1016/j.bpj.2015.05.014. 103 (104) Greenleaf, W. J.; Woodside, M. T.; Abbondanzieri, E. A.; Block, S. M. Passive All-Optical Force Clamp for High-Resolution Laser Trapping. Phys. Rev. Lett. 2005, 95 (20), 208102. https://doi.org/10.1103/PhysRevLett.95.208102. (105) Farré, A.; Marsà , F.; Montes-Usategui, M. Optimized Back-Focal-Plane Interferometry Directly Measures Forces of Optically Trapped Particles. Opt. Express 2012, 20 (11), 12270â12291. https://doi.org/10.1364/OE.20.012270. (106) Allersma, M. W.; Gittes, F.; deCastro, M. J.; Stewart, R. J.; Schmidt, C. F. Two-Dimensional Tracking of Ncd Motility by Back Focal Plane Interferometry. Biophys. J. 1998, 74 (2), 1074â1085. https://doi.org/10.1016/S0006-3495(98)74031-7. (107) Mahamdeh, M. HIGH RESOLUTION OPTICAL TWEEZERS OPTIMIZED FOR BIOLOGICAL STUDIES. (108) Capitanio, M.; Romano, G.; Ballerini, R.; Giuntini, M.; Pavone, F. S.; Dunlap, D.; Finzi, L. Calibration of Optical Tweezers with Differential Interference Contrast Signals. Rev. Sci. Instrum. 2002, 73 (4), 1687â1696. https://doi.org/10.1063/1.1460929. (109) Cronin-Golomb, M.; Domachuk, P.; Mägi, E. C.; Perry, H.; Omenetto, F.; Eggleton, B. J. Applications of Optical Tweezers to Optofluidics; Psaltis, D., Fainman, Y., Eds.; San Diego, California, USA, 2006; p 63290B. https://doi.org/10.1117/12.679856. (110) Bennink, M. L.; Leuba, S. H.; Leno, G. H.; Zlatanova, J.; de Grooth, B. G.; Greve, J. Unfolding Individual Nucleosomes by Stretching Single Chromatin Fibers with Optical Tweezers. Nat. Struct. Biol. 2001, 8 (7), 606â610. https://doi.org/10.1038/89646. (111) Andersson, M.; Czerwinski, F.; Oddershede, L. B. Optimizing Active and Passive Calibration of Optical Tweezers. J. Opt. 2011, 13 (4), 044020. https://doi.org/10.1088/2040-8978/13/4/044020. (112) Jeyachandran, Y. L.; Mielczarski, J. A.; Mielczarski, E.; Rai, B. Efficiency of Blocking of Non-Specific Interaction of Different Proteins by BSA Adsorbed on Hydrophobic and Hydrophilic Surfaces. J. Colloid Interface Sci. 2010, 341 (1), 136â142. https://doi.org/10.1016/j.jcis.2009.09.007. 104 (113) Dong, J.; Castro, C. E.; Boyce, M. C.; Lang, M. J.; Lindquist, S. Optical Trapping with High Forces Reveals Unexpected Behaviors of Prion Fibrils. Nat. Struct. Mol. Biol. 2010, 17 (12), 1422â1430. https://doi.org/10.1038/nsmb.1954. (114) Roy, R.; Hohng, S.; Ha, T. A Practical Guide to Single-Molecule FRET. Nat. Methods 2008, 5 (6), 507â516. https://doi.org/10.1038/nmeth.1208. (115) Selhuber-Unkel, C.; Nielsen, P.; Berg-Sørensen, K.; Oddershede, L. Variety in Intracellular Diffusion during the Cell Cycle. Phys. Biol. 2009, 6, 025015. https://doi.org/10.1088/1478-3975/6/2/025015. (116) Konyshev, I.; Byvalov, A.; Ananchenko, B.; Fakhrullin, R.; Danilushkina, A.; Dudina, L. Force Interactions between Yersiniae Lipopolysaccharides and Monoclonal Antibodies: An Optical Tweezers Study. J. Biomech. 2020, 99, 109504. https://doi.org/10.1016/j.jbiomech.2019.109504. (117) Chakraborty, A.; Meng, C. A.; Block, S. M. Observing Single RNA Polymerase Molecules Down to Base-Pair Resolution. In Optical Tweezers: Methods and Protocols; Gennerich, A., Ed.; Methods in Molecular Biology; Springer: New York, NY, 2017; pp 391â409. https://doi.org/10.1007/978-1-4939-6421-5_15. (118) Aitken, C. E.; Marshall, R. A.; Puglisi, J. D. An Oxygen Scavenging System for Improvement of Dye Stability in Single-Molecule Fluorescence Experiments. Biophys. J. 2008, 94 (5), 1826â1835. https://doi.org/10.1529/biophysj.107.117689. (119) Duan, J.; Wang, Q.; Wang, Y.-P. HOPS: A Fast Algorithm for Segmenting Piecewise Polynomials of Arbitrary Orders. IEEE Access 2021, 9, 155977â155987. https://doi.org/10.1109/ACCESS.2021.3128902. (120) Drew, H. R.; Wing, R. M.; Takano, T.; Broka, C.; Tanaka, S.; Itakura, K.; Dickerson, R. E. Structure of a B-DNA Dodecamer: Conformation and Dynamics. Proc. Natl. Acad. Sci. U. S. A. 1981, 78 (4), 2179â2183. (121) Neidle, S.; Pearl, L. H.; Skelly, J. V. DNA Structure and Perturbation by Drug Binding. Biochem. J. 1987, 243 (1), 1â13. 105 (122) Rivetti, C.; Codeluppi, S. Accurate Length Determination of DNA Molecules Visualized by Atomic Force Microscopy: Evidence for a Partial B- to A-Form Transition on Mica. Ultramicroscopy 2001, 87 (1), 55â66. https://doi.org/10.1016/S0304-3991(00)00064-4. (123) Rivetti, C.; Guthold, M.; Bustamante, C. Scanning Force Microscopy of DNA Deposited onto Mica: EquilibrationversusKinetic Trapping Studied by Statistical Polymer Chain Analysis. J. Mol. Biol. 1996, 264 (5), 919â932. https://doi.org/10.1006/jmbi.1996.0687. (124) Sanchez-Sevilla, A.; Thimonier, J.; Marilley, M.; Rocca-Serra, J.; Barbet, J. Accuracy of AFM Measurements of the Contour Length of DNA Fragments Adsorbed on Mica in Air and in Aqueous Buffer. Ultramicroscopy 2002, 92 (3â4), 151â158. https://doi.org/10.1016/S0304-3991(02)00128-6. (125) Sakata-Sogawa, K.; Kurachi, M.; Sogawa, K.; Fujii-Kuriyama, Y.; Tashiro, H. Direct Measurement of DNA Molecular Length in Solution Using Optical Tweezers: Detection of Looping Due to Binding Protein Interactions. Eur. Biophys. J. 1998, 27 (1), 55â61. https://doi.org/10.1007/s002490050110. (126) Ojala, H.; Hanhijärvi, K.; Wallin, A.; Ziedaite, G.; Bamford, D.; Hæggström, E. Active Force Clamp Control of Optical Tweezers; 2010; Vol. 98. https://doi.org/10.1016/j.bpj.2009.12.3228. 106 APPENDICES A. Instrument component list (excluding common optomechanical parts such as optical posts, clamp and mount and microfluidic components). Stage Component Model Manufacturer 1 1064nm CW laser V-GEN VCFL-20000 Spectra-Physics [T1] PCX lens (F = 60mm) LA1134-C Thorlabs [T1] PCX lens (F = 38.1mm) KPX079AR.35 Newport Plate Polarizer PSBW 1064 Thorlabs Beam Dumps (x2) LB2 Thorlabs Faraday Isolator ISO-FRDY-05-N Newport Half-wave plate (x2) 05RP02-34 Newport Cube Beamsplitter 05BC16PC.9 Newport 2 Cube Beamsplitter (x3)* L10BC16PC.9 Newport Laser Line Mirror (x2)* 10D20DM.10 Newport Piezo Steering Mirror Mount PSM2SG Newport Piezo Stack Amplifier/Controller NPC3SG Newport [T2] PCX lens (F = 125mm) LA4236-1064 Thorlabs [T2] PCX lens (F = 200mm) KPX106AR.33 Newport 3 Dichroic Mirror (x2) M254H45 Thorlabs Objective lens (M= 60x) UPLSAPO60XW Olympus XYZ Stage Motor (x3) Z825B Thorlabs XYZ Stage Motor Controller (x3) KDC101 Thorlabs Syringe Pump (x2) Pump 11 Elite Havard Apparatus Relay lens (x2) KBX070AR Newport Line Filter 10LF25-1064 Newport Quadrant Photodiodes (x2) QP154-Q-HVSD First Sensor 107 Stage Component Model Manufacturer Visualization LED (λ = 639nm) LED630E Thorlabs LED mount LEDMT1E Thorlabs PCX lens LA1417-A Thorlabs ND filter wheel FHW1ZG Newport ND filters Model 5248 Newport Relay lens LA1433-B Thorlabs Low-Pass Filter FES0950 Thorlabs CMOS camera DCC1645C Thorlabs Data Collection Data Acquisition card (DAQ) USB-6212 National Instrument * Components which were used across multiple stages 108 B. LabVIEW general schematic Block diagram of data acquisition program OT-DAQ/SG Data processing MATLAB script is available for internal on Github: https://github.com/UBC-SMMB/Optical-Tweezers. Please contact Dr. Isaac T.S Li at isaac.li@ubc.ca for any external use (MATLAB and LabVIEW) request. 109 Block diagram of stage motor control program OT-MSC 110 C. Troubleshooting table (chronological order, starting from experiment preparation) Observations Troubleshoot Parafilm cut-out deformed when removing the trimmings CNC cutting blade is blunt, or the film was not properly fixed onto the cutting mat. Replace the blade, adjust the cutting depth and ensure press the film firmly onto the mat. Bubbles trapped/ tearing in sealed area of the flow cell Improper clamping and/or sealing temperature is too low. Up the sealing temperature or prolong the sealing time. Flow cell stuck to oven rack or clamping slide Improper placement of coverglass sandwich. Align the coverglass carefully and trim exposed parafilm to prevent it from melting outside the flow cell. Channel deformed after sealing protocol Sealing temperature is too high or the clamp is too tight. Reduce oven temperature and sealing time while using less clamping clips. Bubbles trapped within microfluidic channels when buffer is introduced Fast buffer injection causes air to be trapped within channel geometry, slow down injection rate, especially during the initial buffer injection. There is also a possibility of contaminated glass surface. Redo the injection by forcing the buffer out using pressurized air Unable to insert buffer into the flow cells Check if the correct side of the chip is facing the inlets. Check for blockage within the inlets and the drilled holes on the flow cell. Water droplets on the inlets drop rapidly This indicates leakage. Inspect the chip, the drilled holes as well as the inlets to make sure that the connection is secure, the glass are not cracked and the parafilm seal is maintained. Bubbles in the syringes or the needle adapters Gently tap on the side to dislodge, stuck bubbles can be dislodged by introduce air into the syringe to merge with these bubbles and gently pushed out. Unable to locate correct microfluidic surface via the camera Move the stage to locate the edge of microfluidic channels (it can appear blurry or simply a slight change in the brightness if the focus is too far off). Visually inspect the location of the chip from the objectives. Ensure that the rear objective is at the correct position (check for laser beam collimation and output beam profile). No particles in the flow channels or at the junction Double check the syringe and its tubing for bubbles and blockage, ensuring that the focusing plane is within the chip. Turn up injection rate to a minimum of 100 µl/hr to fill the channels with particle. Additionally, the particle concentration may be too low or the particles might have lost during washing step. Consider increasing the stock concentration of particles and increasing centrifugation speed. 111 Observations Troubleshoot Particles flows abnormally This indicates problem within microfluidic circuit. Known issues are: (1) the loosening of inlet connections, check for buffer accumulation surrounding the inlets (2) blockage of waste reservoir, especially the air vent hole (3) blockage of the syringes, check for pressure buildup within the needle adapter (4) leakage due to cracked chip surface, especially near the drilled holes where the inlets makes physical contact (5) leakage due to improper sealing of the flow cell (6) bubbles within the system, visually inspect the tubing the microfluidic channels, syringes and waste reservoir. Bubbles formed on the wall of the microfluidic channels during experiment This can be mitigated by injecting wash buffer into the flow cell after it is assembled, allowing the bubbles to form on the sidewalls and simply force them out by introducing a fast flow of buffer and/or repeatedly withdraw and inject until the bubbles are dislodged. Unable to locate single particles Generally, the mixing action during particle preparation phase should be enough to de-aggregate the particle. Additional 1-minute sonication steps may alleviate this problem. This can also solve low particle density problem. Unable to trap particles Check beam profile, there are chances that the objectives are not fully immersed in water. Additionally, the sum voltage on the QPD will reveal any abnormalities in beam path. Lastly, there is also a possibility that the focusing plane is not aligned to the trapping plane. Particles constantly dislodge from trap when stage is moved Trapping force is too weak. Check laser power using power meter and QPD sum voltage, check for beam clipping at the objective aperture and previous stage. Ensure the adjustment ring on the objective is set to account the thickness of the coverglass. Lastly, ensure that the particle is not too close to the surface as it may contact the surface and fly off when the stage is moved. Sudden illumination changes in live camera view This often comes with a noticeable drop in QPD voltage. This is due to the evaporation of water interface between the chip and the objectives front glass. Refilling the interface with Milli-Q water will immediately solve this problem. If problem persist, check for air bubbles. Offsetting trap outside of limit range In rare occasions when the trap needs to be leveled outside of the specified range (10V) or the user requires additional steering range. The steering trap can be manually offset by tuning the adjustment knob of the cube beamsplitter in stage 2 (second one, recombining the polarised beams to the same path). Constantly monitoring the relative beam position to ensure that both beams stay within proximity. 112 Observations Troubleshoot Particles flows in middle channel Check flow ratio (typically 1:3 side channels: middle channel), users may increase the middle channels flow upon their discretion. Furthermore, temporarily stop all flow and observe particle diffusion behaviour will reveal problems within the flow circuit. Reset the flow by introducing a high flow rate of 1000 µl/hr for all channel. Particles escape traps during fishing protocol There may have been too many interactions between the trapped particles. Change the fishing signal to reflect this observation by increasing the dwelling distance will reduce the number of interactions formed. Lowering the coating density will also helps alleviate this problem. In contrast, if the trapped particles escaped when they approach one another, the traps might have been too close to one another that the particles are making physical contacts and pushing each other away from their respective trap centers. No interaction between trapped particles Ensure correct particles are trapped. With a system with multiple particle populations, trapping operation should happen close to the opening of the channel. A flow reset (ie. Introducing high flow of 1000 µl/hr) will eliminate all mix-up. Ensure that particle with longer handle should stay upstream. Increase dwelling time and carefully decrease dwelling distance should be considered. Otherwise, assess functional coating of the particles. Abnormal trapped particle profile This indicates the presence of multiple particles within the trap. Simply discard the current trapped particle and restart the trapping operation. If the particle possesses a non-spherical shape or abnormal âtailâ, impurities may have been introduced within the system. Particle washing step, the buffers and microfluidic components (syringe tubing, inlets and waste reservoir) should be thoroughly inspected. OT-DAQ/SG slow and laggy response Plot buffer may have been set too high, reduce to a value that is below 10x sampling rate. Additionally, restart the acquisition program. OT-MSC unresponsive Occasionally, the communication between the program and motor controller (KDC101, Thorlabs) failed which result in no input being performed. Simply perform reset on both controller and the program. Users will have will need to remove the flow assembly to recalibrate the stage. Shutter digital control unresponsive The shutter controller may have been switched to manual mode. Re-engage modulating mode by turning shutter controller on and hold the ENABLE button until the green LED flashed. If the problem persists, check its connection with the DAQ card and restart the program. Contact program developer if necessary.
Thesis/Dissertation
eng
Biochemistry and Molecular Biology
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Li, Isaac T. S.
Construction of dual-beam optical tweezer for single-molecule study
Text