A fluorogenic array for temporally unlimited single-molecule tracking


We describe three optical tags, ArrayG, ArrayD and ArrayG/N, for intracellular tracking of single molecules over milliseconds to hours. ArrayG is a fluorogenic tag composed of a green fluorescent protein–nanobody array and monomeric wild-type green fluorescent protein binders that are initially dim but brighten ~26-fold on binding with the array. By balancing the rates of binder production, photobleaching and stochastic binder exchange, we achieve temporally unlimited tracking of single molecules. High-speed tracking of ArrayG-tagged kinesins and integrins for thousands of frames reveals novel dynamical features. Tracking of single histones at 0.5 Hz for >1 hour with the import competent ArrayG/N tag shows that chromosomal loci behave as Rouse polymers with visco-elastic memory and exhibit a non-Gaussian displacement distribution. ArrayD, based on a dihydrofolate reductase nanobody array and dihydrofolate reductase–fluorophore binder, enables dual-color imaging. The arrays combine brightness, fluorogenicity, fluorescence replenishment and extended fluorophore choice, opening new avenues for tracking single molecules in living cells.

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Fig. 1: A dynamic recruitment-based approach for prolonged imaging and to abolish aggregation propensity.
Fig. 2: Fluorescence enhancement of mwtGFP on binding with the GBP1 array suppresses background fluorescence.
Fig. 3: Hour-duration tracking of single histones in the nucleus using ArrayG/N.
Fig. 4: Kinesin-ArrayG dynamics.
Fig. 5: Evaluation of integrin β1-ArrayG16× functionality.
Fig. 6: Hidden Markov trajectory segmentation using vbSPT.

Data availability

All datasets and analysis software are available upon request. All plasmid sequences have been submitted to NCBI GenBank and have been assigned the following accession numbers: MK317910, MK317911, MK317912, MK317913, MK317914, MK317915, MK317916, MK317917, MK317918, MK317919, MK317920. Plasmids are available upon request.


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This work was partially supported by the National Institutes of Health, National Institute Of General Medical Sciences/National Cancer Institute (NCI) grant no. GM77856, NCI Physical Sciences Oncology Center grant no. U54CA143836, National Science Foundation Graduate Fellowship Program no. DGE-114747, National Institute Of Biomedical Imaging and Bioengineering/4D Nucleome Roadmap Initiative no. 1U01EB021237, National Institutes of Health Training Grant T32GM008294, National Science Foundation Graduate Fellowship Program no DGE-1656518 and National Science Foundation (NSF), Physics of Living Systems Program (PHY-1707751).

Author information




R.P.G., J.M.F., W.D. and J.T.L. designed the research. R.P.G. and W.D. did most of the cloning. R.P.G generated and optimized most of the cell lines. R.P.G. and J.M.F carried out most of the imaging. W.D. set up the confocal calibration and did the HiLoTIRFM GFP counting. R.P.G and Q.S. carried out the nuclear sequestration experiments. Q.S performed the flow sorting experiments. J.M.F., W.D., Q.S. and R.P.G. analyzed most of the data. A.J.S. and B.B. analyzed the multiscale chromatin dynamics data. R.P.G., J.M.F. and J.T.L. wrote the paper.

Corresponding author

Correspondence to Jan T. Liphardt.

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Supplementary information

Supplementary Information

Supplementary Figures 1–13, Supplementary Note, Supplementary Tables 1–3

Reporting Summary

Supplementary Video 1

Laser-scanning confocal movie of dynamic droplet-like behavior of KIF560-ArrayG aggregates in presence of eGFP.

Supplementary Video 2

TIRF movie of integrin β1-ArrayG16x stably expressed in pKOαVβ1 cell line co-expressing mwtGFP.

Supplementary Video 3

20 Hz prolonged tracking of integrin β1-ArrayG16x + mwtGFP.

Supplementary Video 4

20 Hz HiLo-TIRF movie of H2B-ArrayGN + mwtGFP (left) and corresponding single-molecule trajectories (right).

Supplementary Video 5

HiLo-TIRF movie of H2B-ArrayGN + mwtGFP imaged under 20 Hz (left) and 0.5 Hz (right) showing gradual signal decay for 20 Hz case and constant signal strength for 0.5 Hz case (total illumination times in both cases are the same).

Supplementary Video 6

20 Hz HiLo-TIRFM movie of KIF560-ArrayG + mwtGFP (left) and corresponding single-molecule trajectories (right).

Supplementary Video 7

80 Hz HiLo-TIRFM movie of KIF560-ArrayG + mwtGFP on labeled tubulin.

Supplementary Video 8

20 Hz HiLo-TIRF movie of KIF560-ArrayD + DHFR-mGFP (left) and corresponding single-molecule trajectories (right).

Supplementary Video 9

20 Hz HiLo-TIRF movie of KIF560-ArrayD + DHFR-mCherry (left) and corresponding single-molecule trajectories (right).

Supplementary Video 10

20 Hz Dual-color TIRF imaging of cells co-expressing KIF560-ArrayG + mwtGFP (green) and KIF560-ArrayD + DHFR-mCherry (red) (left) with corresponding single molecule trajectories (right).

Supplementary Video 11

High temporal resolution (180 Hz) tracking of single KIF560-ArrayG molecules, using HiLo-TIRFM.

Supplementary Video 12

20 Hz TIRF imaging of vinculin-mCherry and β1-ArrayG16x + mwtGFP (left) with corresponding single-molecule trajectories (right).

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Ghosh, R.P., Franklin, J.M., Draper, W.E. et al. A fluorogenic array for temporally unlimited single-molecule tracking. Nat Chem Biol 15, 401–409 (2019). https://doi.org/10.1038/s41589-019-0241-6

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