PhotoGate microscopy to track single molecules in crowded environments

Vladislav Belyy, Sheng-Min Shih, Jigar Bandaria, Yongjian Huang, Rosalie E. Lawrence, Roberto Zoncu and Ahmet Yildiz ()
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Vladislav Belyy: Biophysics Graduate Group, University of California
Sheng-Min Shih: University of California
Jigar Bandaria: University of California
Yongjian Huang: Biophysics Graduate Group, University of California
Rosalie E. Lawrence: University of California
Roberto Zoncu: University of California
Ahmet Yildiz: Biophysics Graduate Group, University of California

Nature Communications, 2017, vol. 8, issue 1, 1-10

Abstract: Abstract Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signalling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density. Here we develop the PhotoGate technique that controls the number of fluorescent particles in a region of interest by repeatedly photobleaching its boundary. PhotoGate bypasses the requirement of photoactivation to track single particles at surface densities two orders of magnitude greater than the single-molecule detection limit. Using this method, we observe ligand-induced dimerization of a receptor tyrosine kinase at the cell surface and directly measure binding and dissociation of signalling molecules from early endosomes in a dense cytoplasm with single-molecule resolution. We additionally develop a numerical simulation suite for rapid quantitative optimization of Photogate experimental conditions. PhotoGate yields longer tracking times and more accurate measurements of complex stoichiometry than existing single-molecule imaging methods.

Date: 2017
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DOI: 10.1038/ncomms13978

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