Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signaling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density in the cellular environment. We developed a photobleaching gate assay, which controls the number of fluorescent particles in a region of interest by repeatedly photobleaching its boundary. Using this method, we tracked single particles at surface densities two orders of magnitude higher than the single-molecule detection limit. We observed ligand-induced dimerization of epidermal growth factor receptors (EGFR) on a live cell membrane. In addition, we tracked individual intraflagellar transport (IFT) trains along the length of a cilium and observed their remodeling at the ciliary tip.
Ahmet Yildiz, "PhotoGate microscopy: tracking single molecules in a cytoplasm
(Conference Presentation)," Proc. SPIE 9714, Single Molecule Spectroscopy and Superresolution Imaging IX, 971409 (Presented at SPIE BiOS: February 13, 2016; Published: 27 April 2016); https://doi.org/10.1117/12.2209394.4848767625001.
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