By tightly focusing a 1064nm laser beam, we can apply piconewton forces to single biological molecules while simultaneously measuring sub-nanometer position and fluctuations at >20 kHz. To appreciate how small the forces applied by molecular motors are, consider that one piconewton is roughly the force of gravitational attraction between two average-sized people standing half a mile apart. Our optical trap is built around a modified Nikon Ti-E microscope and incorporates a custom z-focus feedback system that effectively eliminates axial drift. The trap is built in an acoustically quiet and temperature-controlled room to minimize vibrational noise factors − when measuring molecular-scale motions, someone simply closing a door on the other end of the hallway could send the detectors off the charts if the instrument is not properly isolated. The trap is fully automated and remotely operated with custom-written LabVIEW software. The position of our trap in the sample image plane is controlled by a pair of acousto-optical deflectors and can be updated every 50 microseconds, which together with the computational speed of a Xilinx FPGA enables us to implement fast force feedback algorithms and perform accurate position detector calibrations. Among other experiments, we use the trap to apply forces to individual dynein motors walking along axonemes, allowing us detailed insight into the stepping behavior and mechanochemistry of the motor. Additionally, we have two TIRF lines coupled into our setup (488nm and 633nm) so that we have the option to observe single fluorophores on a highly sensitive Andor EMCCD camera.
Figure 1. Reflection of a well-aligned trapping laser off the bottom surface of the coverslip.
Figure 2. Differential detection stage. Splitting the trapping laser beam into orthogonal polarizations allows us to create two traps simultaneously and detect the positions of two trapped beads independently from one another. Differential measurement experiments allow us to de-couple the observed signal from noise sources common to both trapping beams, such as vibrations of the microscope body or stage drift.
Figure 3. Sample stall of a single dynein motor. The motor pulls the bead out of the trap until the restoring force of the trap balances the forward force of the motor, at which point the motor pauses and lingers for a while until it detaches from the microtubule and the bead snaps back to the center of the trap.