Figure 1.

Instrumentation and concept of the tFCS assay. (A) Key optical and electronic elements of the tFCS microscope are shown. The feedback is applied by an analog controller (blue box), which drives the microscope piezo stage and the acousto-optic deflectors (AODs) controlling the position of the tracking and probe beams. The optical elements used to dither the tracking laser axially are omitted for simplification (Supporting Materials and Methods). (B) A schematic of the tracking and probe beam motions during tracking of a freely diffusing molecule is shown (blue). The trajectory (purple line) of the green-labeled site on the molecule (green star, reference dye) is followed by the tracking laser (green beam), whereas the probe laser (red beam) is maintained at a fixed position with respect to the tracking laser. The green ring indicates circular dithering of the tracking beam used to locate the molecule in XY (13). The focus of the rotating tracking laser is also axially dithered between two transverse planes to locate the molecule along the z axis (bottom right) (13). The green-labeled site is immobile with respect to the probe laser (at the tracking/reference lock point, dashed crosshair), whereas the red-labeled site (red star, probe dye) moves because of intramolecular dynamics. (C) Single-molecule signals recorded in tFCS are shown, including the probe fluorescence (red), the stage trajectory, and the tracking laser power (black), which is under feedback control to keep the tracking dye fluorescence at a fixed value (green). (D) A single-molecule fluorescence correlation function $g_p\left(\tau \right)$ from the probe signal in (C) is shown. To see this figure in color, go online.