Fig. 1.

Simultaneous measurements of the position of a fluorescent particle by using AFM and TIRF microcopy. (A) An exponentially decaying evanescent wave field results when a laser beam is reflected at an angle (θ > critical angle, θ_c) across the boundary between two media with different refractive indexes (n₁ > n₂). The evanescent wave transfer function that relates fluorescence intensity and distance is given by I_z = I_o·e^–z/d_p, where I_z is the fluorescence intensity measured at a vertical distance z from the interface, I_o is the intensity measured at the interface, and d_p is the penetration depth. (B) We use an AFM head (Dimension 3100, Veeco Metrology Group) mounted on top of a through-the-lens TIRF microscope (IX71, Olympus) equipped with an EMCCD camera (iXon DV887, Andor Technology) to confirm the exponential relationship between intensity and distance and to directly measure d_p. (C) Simultaneous measurements of fluorescence intensity (upper trace) and cantilever deflection (lower trace) as a fluorescently labeled AFM cantilever is driven in and out of the evanescent wave field. Fluorescence was recorded with an exposure time of 30 ms.