3D Submillisecond tracking microscopy of single fluorescent particles with adaptive optics

Single particle tracking microscopy in combination with fluorescent labeling has opened the door to investigations of nanoscale dynamics in living cells. While conventional instruments feature temporal resolutions of typically 5–30 ms, nanoscale processes happen on a millisecond or submillisecond time scale. To overcome this limitation, I have developed a single particle tracking microscope with 130 μs temporal resolution and single-fluorophore sensitivity. The instrument acquires 3D trajectories by active tracking of a fluorescent particle with a focused laser beam. This is accomplished by fast beam steering, which is feedback-driven by the detected particle position in the focal volume. For translation of the laser focus along the optical axis, I have implemented a novel vibration-free remote focusing mechanism based on a deformable mirror, an adaptive optics wavefront correction device. In characterization experiments with fluorescent beads, I have found that the instrument is capable of tracking directed motion up to 150 μm/s and free 3D Brownian motion with diffusion coefficients of more than 2 μm²/s. The potential for biological applications is demonstrated by tracking fluorescently labeled viruses on cell membranes and transport vesicles in the cytoplasm of living cells