BPS2026 – Real-time visualization and analysis of fibrin polymerization

To form a blood clot, fibrinogen is converted into fibrin through the action of the enzyme thrombin. Fibrin then polymerizes longitudinally and laterally as it matures into a fiber. Polymerization results in a dense, 3-dimensional branched network. Previous research has shown the relevance of these fibrin gel structures in hemostatic conditions; however, the mechanism by which they form has not been fully resolved. Using light sheet microscopy, 3-dimensional volumes of the fibrin polymerization process were captured. Manual annotation of these microscopy videos revealed that fiber branch points occur through the collision and the binding of diffusing fibers rather than through the splitting of growing fiber termini. However, the density of fibers and amount of data greatly slows manual annotation-based analysis and limits the ability to capture important data, such as growth rates and fiber stiffness. To more quickly process these data, a computational approach was utilized. A custom tracking pipeline, suited to the networks formed by cylindrical fibrin fibers, was developed, beginning with an AI-based classifier. This custom pipeline allowed for the tracking of uniquely labeled fibers over time. Automated merge detection between linking phases further improved accuracy. Additionally, network formation was analyzed through skeletonization techniques to measure the number of branches per junction over time. Combining the skeletonization and tracking methods, single fibers were identified by their lack of branch points and tracked. The addition of branch points to previously tracked objects served as a signal for merge detection. This approach yielded measurements of single fibrin fiber diffusion rates, as well as the first volumetric and length growth rates of fibers throughout polymerization. In addition, the gel point was quantified by analyzing the span of connected objects to characterize the network consolidation over time at the level of single fibers.