A fluorogenic nanobody array tag for prolonged single molecule imaging in live cells

Prolonged single molecule imaging in live cells requires labels that do not aggregate, have high contrast, and are photo-stable. To address these requirements, we have generated arrays of modular protein domains that function as fluorophore recruitment platforms. ArrayG, a linear repeat of GFP-nanobodies, recruits free monomeric wild-type GFP, which brightens ~15-fold upon binding the array. The fluorogenic ArrayG tag effectively eliminates background fluorescence from free binders, a major impediment to high-throughput acquisition of long trajectories in recruitment based imaging strategies. The photo-stability of ArrayG and consistently low background made it possible to continuously track single integrins for as long as 105 seconds (2100 frames). Prolonged tracking of both kinesin and integrin revealed repeated state-switching events, a measurement capability that is crucial to a mechanistic understanding of complex cellular processes. We also report an orthogonal array tag, based on a DHFR-nanobody, for prolonged dual color imaging of single molecules.