Real-time 3D Single Molecule Tracking
To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of "locking on" to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART was applied to actively track single Atto 647N fluorophores in 90% glycerol solution with an average duration of ~16 s at count rates of ~10 kHz. Active feedback tracking was further applied to single proteins and nucleic acids, directly measuring the diffusion coefficient of various lengths (99 to 1385 bp) of single DNA molecules at speeds up to 10 m2/s. In addition, 3D-SMART was able to quantify to occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.