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. Author manuscript; available in PMC: 2018 May 4.
Published in final edited form as: Nanoscale. 2017 May 4;9(17):5664–5670. doi: 10.1039/c7nr01369h

Figure 1.

Figure 1

Schematic of our solution-based, spatiotemporally resolved single DNA annealing-melting kinetics measurement method. 3D single-molecule tracking is achieved by a confocal-feedback scheme (Top left). Active feedback provided by an xyz piezo stage is employed to keep the DNA molecule at the center of the excitation focus, where the motion history of the piezo stage represents the 3D trajectory of the tracked molecule (Bottom left). The time-tagged-time-resolved (TTTR) mode of the TCSPC module allows us to monitor the change of reporter’s fluorescence lifetime at 15-ms temporal resolution. In our DNA model system, the longer lifetime (from unquenched dye) represents the ssDNA state (red segment) while the shorter lifetime (from quenched dye) indicates the dsDNA state (blue segment) (Top right). The acquired single-molecule lifetime trace is mapped onto the molecule’s 3D trajectory, providing not only the temporal but also the spatial information of annealing-melting events that take place along this 1,015-ms trajectory (Bottom right).