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Ot practicle guide A Practical Guide to Optical Trapping Joshua W Shaevitz jshaevitz berkeley edu October C CChapter Introduction to optical trapping In the last few decades novel microscopy techniques have been developed to monitor the activity of single

A Practical Guide to Optical Trapping Joshua W Shaevitz jshaevitz berkeley edu October C CChapter Introduction to optical trapping In the last few decades novel microscopy techniques have been developed to monitor the activity of single enzymes as they perform their biological functions in vitro Motor proteins such as kinesin myosin F Fo ATPase and RNA polymerase have been mercilessly subjected to magnetic elastic and optical forces In Ashkin and colleagues reported the ?rst observation of a stable three-dimensional optical trap or optical tweezers created using radiation pressure from a single laser beam Only a few years later Block and colleagues had used an optical trap to manipulate and apply forces to E coli agella and single kinesin motors Optical traps use light to manipulate microscopic objects as small as nm using the radiation pressure from a focused laser beam In addition measurement of the light de ection yields information about the position of the object in the laser focus Many excellent reviews have been written about optical trapping its uses and designs see e g In particular Lang and Block is a thorough review of the optical trapping literature This manuscript is meant to be a practical guide to understanding optical traps and not an in depth review When possible simple examples and explanations are used to give the reader an intuitive feel for how these systems work and how they are implemented I hope that this document will continue to improve and welcome any comments The picoNewton and nanometer ranges of force and distance accessible to optical traps make them particularly useful for studying biological systems Fig Optical forces have been used to investigate structural properties of biological polymers such as DNA membranes whole cells and microtubules Microrheological properties of these objects can be probed through the application of forces either to the object itself or to a small dielectric sphere or bead to which the object is attached Molecular motors represent the most used application of optical traps in the biological sciences A great deal has been learned about kinesin dynein C CHAPTER INTRODUCTION TO OPTICAL TRAPPING myosin and RNA polymerase using optical forces C Figure Di ?erent Optical Trapping Assays A Optical trapping studies of RNA polymerase typically ?x the polymerase to a optically trapped bead while the distal end of the DNA is attached to the microscope coverslip As the polymerase moves along the DNA it must do work against the optical trap B In a typical assay of kinesin motion a motor ?? coated bead moves along a microtubule towards its plus ??end while being subjected to a retarding force by the optical trap C Studies of non-processive motors such as muscle myosin and NCD often involve more complicated geometries and multiple optical traps In this study a myosin motor eetingly grabs the actin ?lament suspended between the two optical trapped beads and strokes before letting go D Optical traps can also be used to study the polymerization of bio ?laments Here a polymerizing