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Dual-Beam Optical Tweezers D 522 Dual-Beam Optical Tweezers Another example demonstrated the ability of dual- Fogarty K, McPhee JT, Scott E, Van Orden A. Probing the ionic beam FCCS to measure the effective charge of DNA atmosphere of single-stranded DNA using continuous flow capillary electrophoresis and fluorescence correlation spec- molecules interacting with various counterions in con- troscopy. Anal Chem. 2009;81:465–72. tinuous flow capillary electrophoresis (Fogarty et al. Jung J, Van Orden A. A three-state mechanism for DNA hairpin 2009). The effective charge, Zeff, of a molecule is folding characterized by multiparameter fluorescence fluctu- ation spectroscopy. J Am Chem Soc. 2006;128:1240–9. related to the electrophoretic mobility, me, and diffu- Jung J, Ihly R, Scott E, Yu M, Van Orden A. Probing the sion constant, D, according to the equation: complete folding trajectory of a DNA hairpin using dual beam fluorescence fluctuation spectroscopy. J Phys m k T Chem B. 2008;112:127–33. Z ¼ e B (3) eff De LeCaptain DJ, Van Orden A. Two-beam fluorescence cross- correlation spectroscopy spectroscopy in an electrophoretic shift assay. Anal Chem. 2002;74:1171–6. Hence, both the electrophoretic mobility and diffu- Schiro PG, Kuyper CL, Chiu DT. Continuous-flow single- sion constant must be known to accurately measure the molecule CE with high efficiency efficiency. Electrophore- effective charge. Dual-beam FCCS was used to accu- sis. 2007;28:2430–8. Van Orden A, Jung J. Fluorescence correlation spectroscopy for rately measure these parameters simultaneously based probing the kinetics and mechanisms of DNA hairpin forma- on its ability to resolve the flow and diffusion proper- tion. Biopolymers. 2008;89:17–6. ties of the molecules. Thus, the interactions of DNA Widengren J, Mets U. Conceptual basis of fluorescence correla- with various counterions can be studied. tion spectroscopy and related techniques as tools in biosci- ence. In: Zander C, Enderlein J, Keller RA, editors. Single In summary, dual-beam FCCS serves an important molecule detection in solution: methods and applications. role within the family of FCS-related techniques due to Berlin: Wiley-VCH; 2002. p. 69–120. its ability to analyze molecular processes taking place in liquid solutions undergoing unidirectional flow. Such techniques will continue to play a role in charac- terizing such biophysical processes as DNA, RNA, and Dual-Beam Optical Tweezers protein binding, biomolecule folding, and the electrostatic processes of biomolecules and their Sivaraj Sivaramakrishnan1, Jong Min Sung2, associated ions. Alexander R. Dunn3 and James A. Spudich4 1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, USA Cross-References 2Departments of Biochemistry and Applied Physics, Stanford University, Stanford, CA, USA ▶ Fluorescence Correlation Spectroscopy 3Department of Chemical Engineering, Stanford ▶ Fluorescence Cross-Correlation Spectroscopy University, Stanford, CA, USA ▶ Fluorescence Fluctuation Spectroscopy 4Department of Biochemistry, Stanford University ▶ Microfluidics for Single Molecule Detection School of Medicine, Stanford, CA, USA ▶ Single Fluorophore Blinking ▶ Single Fluorophores Photobleaching Synonyms References Molecular motors; Optical trap; Optical tweezers; Brinkmeier M. Cross-correlated flow analysis in microstruc- Single-molecule methods tures. In: Rigler R, Elson ES, editors. Fluorescence correla- tion spectroscopy: theory and applications. Berlin: Springer; 2001. p. 379–95. Fogarty K, Van Orden A. Two-beam fluorescence Definition cross-correlation spectroscopy for simultaneous analysis of positive and negative ions in continuous-flow capillary elec- An optical trap is a device that uses a focused laser trophoresis. Anal Chem. 2003;75:6634–41. beam to create a potential well, in which a small object Fogarty K, Van Orden A. Fluorescence correlation spectroscopy for ultra-sensitive DNA analysis in continuous flow capillary (100 nm to a few micrometers) whose refractive index electrophoresis. Methods. 2009;47:151–8. is greater than that of the surrounding fluid can be Dual-Beam Optical Tweezers 523 D trapped. The object experiences a restoring force that is Svoboda and Block (Svoboda and Block 1994). directed toward the center of the potential well, which The specific layout of the optical trap elements is is directly proportional to the distance of the object dependent on the biological question. In this entry, from the center of the potential well. An optical trap the details of a dual-beam optical trap are discussed, can be used to apply piconewton forces to biological based on the three-bead assay to study myosin marcomolecules such as proteins and DNA. A dual- molecular motors and originally developed by Finer beam optical tweezer consists of two focused laser et al. (1994); for advancements see Sung et al. (2010). beams that are used to trap two different objects and move them independently in solution. A common bio- D logical application of the dual-beam optical tweezer is Myosin Function Studied Using a Dual-Beam the measurement of the biophysical properties of Optical Trap molecular motors. The broad goal of this instrument is to investigate the coupling between protein structure and function. In Basic Concept of an Optical Trap this entry, the setup of a dual-beam optical tweezer as it relates to investigating the mechanical stroke and Light has momentum and a force is generated when the the chemical ATPase cycle of the myosin motor is momentum is changed. When light passes through an discussed. A detailed description of the setup, align- interface between two different media, reflection and ment, and calibration of different optical components refraction occur at the interface. Consider the example can be found in Sung et al. (2010). With minor modi- of a laser beam that is focused at the center of a fications, the dual-beam optical trap setup can be spherical object that has a higher refractive index (n) applied to a variety of biological applications. The than that of the surrounding aqueous medium. Reflec- dual-beam optical trap has been used to study the tion of light at the surface of the object generates workings of myosin at the single molecule level, with a force (radiation force) on the object, in the direction high spatial (nm) and temporal (ms) resolution of light propagation, with a magnitude proportional to (Finer et al. 1994). Figure 1 shows the geometric the beam power. Refraction of light, on the other hand, configuration of different elements to investigate the produces a force (gradient force) that attracts the object interaction between a single actin filament and a single to the focal point of the laser beam, with a magnitude myosin molecule. A single biotinylated actin filament proportional to the gradient of beam power. The object is attached at each end to a 1 mm polystyrene bead, is “trapped” at a point where the vector sum of the through a neutravidin-biotin linkage. The two polysty- gradient and radiation forces is zero (Ashkin 1992). rene beads are held in two independent optical traps. This point is referred to as the center of the trap and is The optical traps are positioned such that the actin located near the focal point of a high numerical aper- filament is stretched taut. Myosin molecules are ture (NA) microscope objective lens (NA > 1.2, and attached to 1.5-mm platform beads using either an most often 1.4). antibody linkage or nonspecific adsorption. The plat- For biological applications, where the optical trap is form bead is moved using the microscope stage such used to apply a force on protein/DNA molecules, the that the actin filament is positioned close to the myosin trapped object is usually a polystyrene (n ¼ 1.57) molecule. Myosin binding to actin results in a stroke of sphere ranging in size from several hundred nanome- its lever arm. Since the myosin is anchored to the ters to a few micrometers. The force experienced by surface, the myosin stroke displaces the actin filament the trapped object increases linearly with distance along its length. This in turn causes a displacement of from the trap center. The strength of the trap is the optically trapped beads at the two ends of the expressed in terms of the “trap stiffness” and varies actin filament. Bead displacement is monitored by for most applications from 0.005 to 1 pN/nm. The force a quadrant photo diode or a position-sensitive detector experienced by the trapped object is therefore the and is a measure of the stroke size of the myosin motor. product of the trap stiffness and distance from Following each stroke, the myosin exchanges ADP for the trap center. A more detailed theoretical description ATP and detaches from the actin filament. For of how the laser traps such particles can be found in processive myosins, each of the two heads alternately D 524 Dual-Beam Optical Tweezers Position sensitive detectors IR laser beam Actin filament Optically trapped bead Myosin GFP antibody Platform bead Coverslip (Not to scale) Dual-Beam Optical Tweezers, Fig. 1 Configuration of acto- A myosin molecule, attached to the platform bead through an myosin in a dual-beam optical trap. An actin filament is attached antibody linkage, interacts with the actin filament. A myosin at each end to a 1-mm polystyrene bead. The polystyrene beads stroke results in the displacement of the actin filament along its are held in two separate optical traps. The actin filament is length. Position-sensitive detectors are calibrated to read out stretched taut and positioned close to a platform bead (1.5 mm). the displacement of the trapped beads binds and releases from the actin filament, resulting in laser is used for detection of the optically trapped beads. a processive movement of the actin filament relative to Similar to the trapping laser, the detection laser is the myosin, which translates into a stepwise movement steered using PMs and is split and recombined using of the optically trapped beads.
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