RT Journal Article SR Electronic T1 Stochastic Protein Labeling Enables Long-term Single Molecule Observation in vivo JF bioRxiv FD Cold Spring Harbor Laboratory SP 116186 DO 10.1101/116186 A1 Hui Liu A1 Peng Dong A1 Maria S. Ioannou A1 Li Li A1 Jamien Shea A1 Amalia Pasolli A1 Jonathan Grimm A1 Pat Rivlin A1 Luke D. Lavis A1 Minoru Koyama A1 Zhe Liu YR 2017 UL http://biorxiv.org/content/early/2017/03/13/116186.abstract AB Our ability to unambiguously image and track individual molecules in live cells is limited by packing of multiple copies of labeled molecules within the resolution limit. Here we devise a universal genetic strategy to precisely control protein copy number in a cell. This system has a dynamic titration range of more than 10,000 fold, enabling sparse labeling of proteins expressed at widely different levels. Combined with fluorescence signal amplification tags, this system extends the duration of automated single-molecule tracking by 2 orders of magnitude. We demonstrate long-term imaging of synaptic vesicle dynamics in cultured neurons as well as in live zebrafish. We found that axon initial segment utilizes a ‘waterfall’ mechanism gating synaptic vesicle transport polarity by promoting anterograde transport processivity. Long-time observation also reveals that transcription factor Sox2 samples clustered binding sites in spatially-restricted sub-nuclear regions, suggesting that topological structures in the nucleus shape local gene activities by a sequestering mechanism. This strategy thus greatly expands the spatiotemporal length scales of live-cell single-molecule measurements for a quantitative understanding of complex control of molecular dynamics in vivo.