Figure 5.

Total internal reflection (TIR) microscopy taps on the production of an evanescent wave, which specifically illuminates biomacromolecules (circles) close to the solid substrate (e.g., fused silica). Immediately after photobleaching, the fluorescent tag on the biomacromolecules were deactivated by the intense laser beam (purple circles) and lost the fluorescence signal to be detected by CCD camera or photomultiplier tube. Afterwards, the fluorescence signal recovers by the exchange of the bleached molecules inside the region of interest with those unbleached biomacromolecules (red circles) through adsorption/desorption kinetics and surface diffusion.