Single dye molecules observed by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy (TIRFM) was used to image single molecules with evanescent waves. The molecules were excited by an evanescent wave with different intensities. Single molecules were imaged on low-noise high-quantum-yield charge-coupled device (CCD) cameras. Two-step photobleaching behavior was observed. Duration from fluorescent spots appearing to disappearing was counted. The duration was decided by the speed of photobleaching. Peak intensity was counted as exciting intensity change. The proportion relationship between the reciprocal of duration and exciting intensity was obtained. The emitted intensity and duration of fluorescein were compared with GFP. A single molecules emit the same number photons was proved.