Application of 3-D Prismatic Optical Tracking to Single-Molecule Optical Tweezers

Abstract

We have developed a new method for tracking single particles in three dimensions under a conventional microscope by dividing light in two components using a wedge prism. This method, three-dimensional prismatic optical tracking, termed tPOT, is here combined with optical tweezers to quantify molecular scale 3-D force in motor proteins. Precise calibration of trapping force to bead displacement with sub nanometer scale enables us to determine the force exerted not only in xy-plane direction but also along optical axis (z-axis). With this experimental set-up, we applied force to rotary motor F1-ATPase that showed rotation on a glass surface. Our research group previously used the procedure in which the objective was displaced to impose the load on single molecules held between the trapped bead and the glass, which frequently caused the measurement error because of drifting motions of the specimen. This method has been modified as follows; 1) the trap center is displaced with time by moving the focusing lens along z-axis; 2) spring constant of the trap is increased via the adjustment of the laser power. In either case positions of the stage and the objective were fixed and thus repetitive measurements were reliably performed. The rupture force of the bead from the glass surface, which presumably reflects the interaction between the shaft and the cylinder of F1-ATPase, distributed up to 40 pN and showed 19 pN on the average. The wide distribution of the force may be originated from different chemical states of the cylinder when the rupture occurs. We also demonstrate various applications of tPOT, such as corkscrewing motions of linear molecular motors or beating motions of ciliary axonemes.