Visualization of the dynamics of fibrin clot growth 1 molecule at a time by total internal reflection fluorescence microscopy.

Individual fluorescently labeled fibrin(ogen) molecules and their assembly to make a clot were observed by total internal reflection fluorescence microscopy (TIRFM). We used the bleaching of the fluorescent labels to determine the number of active fluorophores attached nonspecifically to each molecule. From the total intensity of bleaching steps, as single-molecule signature events, and the distribution of active labeling, we developed a new single-molecule intensity calibration, which accounts for all molecules, including those “not seen.” Live observation of fibrin polymerization in TIRFM by diffusive mixing of thrombin and plasma revealed the real-time growth kinetics of individual fibrin fibers quantitatively at the molecular level. Some fibers thickened in time to thousands of molecules across, equivalent to hundreds of nanometers in diameter, whereas others reached an early stationary state at smaller diameters. This new approach to determine the molecular dynamics of fiber growth provides information important for understanding clotting mechanisms and the associated clinical implications.