This chapter discusses methods used for tracking nanometer movements of single motor molecules with high precision. General principles are described. Approaches based on video are most convenient; however, improved temporal resolution and spatial sensitivity are achieved by custom photodiode detection systems with custom illumination. In the case of microtubules gliding on motor-coated glass, translocation while pivoting around a nodal point is a strong indicator of motility by a single motor molecule. On the other hand, in the case of bead movements statistical arguments have been used to show that the movements observed are driven by single motors. The use of the optical tweezers in those assays is important to make an unbiased sample of the population of beads. To record the position of particles—moving by single motors—requires a major commitment of resources, either to a specialized system or to a high-quality video-enhanced differential interference contrast (DIC) microscope. The lack of image formation in the photodiode systems is compensated by their greater temporal and spatial resolution. However, in contrast, the video-enhanced DIC systems have lower resolution and give additional information about the microtubule position and allow the use of fiducial marker beads in the system.
ASJC Scopus subject areas
- Cell Biology