FIGURE 1.

Total internal reflection fluorescence imaging of purified monomeric GFP. A, representative frames from an image sequence of GFP bleaching with individual GFP molecules highlighted by colored circles. Times shown are relative to the initial image. Data were acquired at 10 Hz using a 100-ms integration time (scale bar,2 μm). B, histogram of averaged background-corrected, area-integrated fluorescence intensities of individual GFP molecules. Resolvable fluorescent spots that remained fluorescent for at least 3 s prior to bleaching were analyzed. C, gallery of photobleaching events showing one-step photobleaching of individual GFP molecules. Arrows correspond to GFP blinking events.