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A Fluorogenic Array Tag for Temporally Unlimited Single Molecule Tracking
- Publication Year :
- 2017
- Publisher :
- Cold Spring Harbor Laboratory, 2017.
-
Abstract
- Cellular processes take place over many timescales, prompting the development of precision measurement technologies that cover milliseconds to hours. Here we describe ArrayG, a bipartite fluorogenic system composed of a GFP-nanobody array and monomeric wtGFP binders. The free binders are initially dim but brighten 15 fold upon binding the array, suppressing background fluorescence. By balancing rates of intracellular binder production, photo-bleaching, and stochastic binder exchange on the array, we achieved temporally unlimited tracking of single molecules. Fast (20-180Hz) tracking of ArrayG tagged kinesins and integrins, for thousands of frames, revealed repeated state-switching and molecular heterogeneity. Slow (0.5 Hz) tracking of single histones for as long as 1 hour showed fractal dynamics of chromatin. We also report ArrayD, a DHFR-nanobody-array tag for dual color imaging. The arrays are aggregation resistant and combine high brightness, background suppression, fluorescence replenishment, and extended choice of fluorophores, opening new avenues for seeing and tracking single molecules in living cells.
- Subjects :
- Physics
Background fluorescence
0303 health sciences
Brightness
Molecular heterogeneity
Fluorescence
03 medical and health sciences
0302 clinical medicine
Background suppression
Molecule
Biological system
Fractal dynamics
Dual color
030217 neurology & neurosurgery
Simulation
030304 developmental biology
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Accession number :
- edsair.doi.dedup.....6cf6b37cdb07ad2b902667fde1f73c3b
- Full Text :
- https://doi.org/10.1101/159004