Untethering Single Molecule Spectroscopy with 3D-SMART

Abstract

Measuring the behavior of single molecules enables the discovery of states and dynamics obscured by bulk measurements. However, molecules in solution rapidly diffuse in three dimensions, precluding long-duration and high-temporal resolution measurement. To overcome this hurdle, we have developed 3D single-molecule active real-time tracking (3D-SMART) which enables active feedback tracking of rapidly diffusing and lowly emitting fluorescent particles. 3D-SMART uses a 3D flying laser focus to tag each arriving photon with 3D position information and a piezoelectric nanopositioner to lock the diffusing object in the objective focal volume, at diffusive speeds exceeding 10 μm2/sec and emission rates of 10 kHz or less. Here we demonstrate the application of 3D-SMART to a range targets, from single virus-like particles and quantum dots in water, all the way down to single fluorophores in viscous solution. This new single molecule tracking can be applied to continuously monitor single proteins and nucleic acids, including real-time measurement of transcription on a freely diffusing, single-dye labelled DNA strand. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.