Bleaching of single fluorescently labeled fibrinogen molecules in TIRFM. (A) TIRFM image showing single fibrinogen molecules that adhered to the chamber glass surface from bulk solution. Time sequences of images like this showed the irreversible bleaching of the attached fluorophores in (B) 1 step, (C) 2 steps, (D) 3 steps, (E) 4 steps, or (F) showed sometimes an exponential decay. (G) Intensities of steps were in the same range for all individual bleaching events. Data plotted are from 1 single set of measurements and all the other datasets present similar values. Each data point represents a mean of at least 5 values and error bars are standard deviations. Because the magnitudes of the bleaching steps are all multiples of a single bleaching step, the number of bleaching steps accounts for the number of fluorophores attached, whereas the exponential decay curve accounts for a larger number of dyes attached to a fibrinogen molecule (number that can be determined from the intensity analysis). (H) Bleaching event distributions for several bulk-labeling ratios using tetramethyl-rhodamine–labeled fibrinogen show that by increasing the bulk-labeling ratio, the 1-step bleaching events decrease in favor of multiple steps events; however, 1-step bleaching events remain predominant. This distribution of active labeling we used for the molecular calibration. At least 90 bleaching events were collected for each ratio. The lines are linear fits of 1-step bleaching events, 2-step bleaching events, and so on. We used an Olympus Plan Apo 60×/1.45 oil TIRFM objective and an ORCA-ER Hamamatsu (Japan) camera. Images were acquired with National Instruments IMAQ Vision Builder 6 and image sequences were recorded at 1 frame/s. Resolution in the TIRFM image was 80 nm/pixel.