Single-channel LFC. (a) Single-channel LFC mounted on the microscope stage and connected to the pumping system. (b) The 3D design of the single-channel LFC. (c) The depiction of immobilization and linearization of a single DNA molecule (green line) interacting with a fluorescently labelled protein (red circle). The DNA is held in place using an optical trap (red cone) to control a polystyrene microsphere (black circle) attached to one end of the DNA. (d) Bright-field microscope image of the trapped polystyrene microsphere (top; position of DNA indicated with dashed line), pseudo-coloured fluorescent image of DNA labelled with YOYO (middle), pseudo-coloured fluorescent image of a single protein molecule labelled with ATTO647N while scanning along the DNA with gamma adjusted to 1.3 (bottom). White scale bars equal 1 µm. (e) A kymograph of the movement of protein along DNA with a duration of around 2 seconds; the horizontal and vertical white scale bars equal 1 µm and 200 ms, respectively. (f) Density distribution of proteins’ movement within frame intervals of 13.5 m for a collection of 49 trajectories of AlkF as exemplified in (e). (g) Mean squared displacement (MSD) of 49 scanning trajectories of AlkF along DNA. Error bars represent standard error of the mean (SEM).