FIGURE 1.

Through-Prism Total Internal Reflection with Continuous Photobleaching (TIR-CP). (a) Schematic of the instrumentation: PMT, photomultiplier tube; BF, barrier filter; DM, dichroic mirror; PP, polarization paper (see Methods); ND, neutral density filter. (b) A fused silica prism is optically coupled with glycerol to a sandwich made from fused silica and microscope slides. (c) Small structures that extend a distance L into the solution are attached to the lower surface of the fused silica slide. A laser beam is internally reflected at the interface of the fused silica slide and the internal solution of the sandwich to create an evanescent field whose intensity decays exponentially with distance, z, from the interface. The characteristic distance of this decay, d, is much smaller than L. Molecules contained within the small structure do not fluoresce (grey circles) until they diffuse into the surface-associated evanescent field. (d) Eventually, the fluorescent molecules (green circles) are permanently bleached (black circles) by exposure to the evanescent field. At low excitation intensities, the decay of evanescently excited fluorescence with time is dominated by the propensity for photobleaching. At high excitation intensities, the decay of fluorescence with time is dominated by diffusion through the length of the small structure.