Fibrin plays a vital role in hemostasis as the fibrous protein network that comprises blood clots and is able to accommodate extremely large tensile strains due to its hierarchical nature. While a multiscale force accommodation mechanism has been proposed, it has been challenging to precisely visualize molecular deformation of fibrin monomers within fibers under tensile loading. Although fibrin molecular structure can be measured with X-ray scattering or vibrational spectroscopy, neither of these tools can image molecular deformation at the scale of individual fibers. Here we introduce a method using intramolecular Förster resonance energy transfer (FRET) by fluorescence lifetime imaging (FLIM) to image and measure the molecular deformation of fibrin at the scale of individual fibers in 3D fibrin networks. FRET-FLIM avoids challenges associated with intensity-based FRET measurements, which can be convoluted by changes in fiber packing under strain that modify the fluorescence intensity but are unrelated to molecular deformation. Fluorescence lifetime overcomes these challenges owing to the independence of the probe concentration as well as the excitation power and allows us to precisely characterize the molecular deformation of fibrin under loading with nanometer resolution.<br/>Based on donor-lifetime measurements, we observed an 18% and 26% decrease in FRET efficiency for 100% and 160% tensile strain, respectively, indicating an increase in the donor-acceptor separation distance. Furthermore, the integrative analysis of fiber orientation and fluorescence lifetime revealed the fibrin strain-bearing mechanism. We find that 100% tensile strain changes the fibrin network orientation from random to a well-aligned state. When increasing tensile strain from 100% to 160%, the fiber orientation and the probe intensity remained the same, but the donor lifetime continued to increase, demonstrating that larger strains are accommodated by fibrin deformation. This result is consistent with our previous work showing secondary structural transitions from α-helices to β-sheets at large strains. Our results show the advantage of using combinatorial donor-lifetime and intensity-based imaging to map molecular deformation at nanometer scales. With the proper experimental design, this FRET-FLIM method can be used to measure local mechanical forces in complete clots (biocomposite materials).

Probing fibrin's molecular response to tensile loading <i>via</i> FRET-FLIM

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Probing fibrin's molecular response to tensile loading via FRET-FLIM