Single molecule spectroscopy across multiple timescales is typically restricted to immobilized proteins on surfaces due to rapid three-dimensional diffusion of unbound probes. A potential solution to "un-tether" surface-bound single molecule methods is real-time 3D single particle tracking (RT-3D-SPT), which uses active feedback to lock onto a freely diffusing probe's position in real-time, enabling continuous observation of dynamic processes with photon-limited temporal resolution. However, RT-3D-SPT methods have yet to translate to real-time single <u>molecule</u> tracking due to the requirement of high emissive rates (&gt;100kHz). This work describes a new advance in RT-3D-SPT called 3D Dynamic Photon Localization Tracking (3D-DyPLoT) which enables active feedback tracking of rapidly diffusing and lowly emitting fluorescent particles. 3D-DyPLoT 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 μm<sup>2</sup>/sec and emission rates of 10 kHz or less. Here we demonstrate the application of 3D-DyPLoT a range targets, from single virus-like particles and quantum dots in water, all the way down to single fluorophores in viscous solution (Fig. 1). 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.

Active feedback tracking of single viruses and fluorophores in solution

2019 Spring National Meeting

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