Quantitative Characterization of Filament Dynamics by Single-Molecule Lifetime Measurements
This chapter discusses some of the current experimental methods used for obtaining quantitative information about single molecules.
The discussion focuses specifically on imaging cytoskeletal molecules- actin and microtubulues, filmanets that can polymerize on one end and depolymerize on the other. Spindles were imaged by first labeling them with a fluorescent dye that attached to the individual tubulin molecules and then imaging using spinning-disk confocal microscopy. Taking movies of the system reveals that the tubulin molecules are in constant movement. To perform data analysis one needs to carry out particle tracking. To do this, particles must first be identified in each time frame, and then must be linked from frame to frame in order to create a trajectory for each particle.
This particular study was looking at the dissapearance and appearance of tubulin molecules. When they appeared, this indicated that a tubulin molecule was incorporated into the spindle, and when one disappeared this meant that the corresponding molecule had been released from the spindle. By tracking how long a tubulin molecule stays in the microtubule for one can measure a 'lifetime' of the particle and thus get information about microtubule function and formation.