R38 - Super-Resolution Mapping of Fibrin Networks Using Expansion Microscopy

Introduction: : Fibrin network is a major structural component of blood clots and thrombi. Structural properties of fibrin networks underly nonlinear mechanical response of clots to stretching, compressive and shear loads, and therefore play an important biological role by providing clot mechanical stability. Common imaging methods, such as fluorescence microscopy and scanning electron microscopy (SEM), face limitations: fluorescence microscopy cannot resolve fibrin fibers thinner than 0.25 μm due to the diffraction limit (~300 nm), making accurate measurements of fiber diameter impossible, and sample preparation for SEM often distorts structures, precluding accurate assessment. To overcome these limitations, we modified and applied expansion microscopy (ExM) to human plasma clots to perform super-resolution imaging of fibrin structures for their enhanced quantification.

Materials and

Methods: : Fibrin network is a major structural component of blood clots and thrombi. Structural properties of fibrin networks underly nonlinear mechanical response of clots to stretching, compressive and shear loads, and therefore play an important biological role by providing clot mechanical stability. Common imaging methods, such as fluorescence microscopy and scanning electron microscopy (SEM), face limitations: fluorescence microscopy cannot resolve fibrin fibers thinner than 0.25 μm due to the diffraction limit (~300 nm), making accurate measurements of fiber diameter impossible, and sample preparation for SEM often distorts structures, precluding accurate assessment. To overcome these limitations, we modified and applied expansion microscopy (ExM) to human plasma clots to perform super-resolution imaging of fibrin structures for their enhanced quantification.

Results, Conclusions, and Discussions:: The hydrogel expansion factor increased nonlinearly over time, reaching up to a 5-fold expansion. Imaging revealed a log-normal distribution of fibrin fiber thickness and a nonlinear, 2.22-fold decrease in mean fiber diameter as thrombin concentration increased from 0.3 to 1.5 U/mL. Measurements of average porosity using ExM images ranged from 0.92 to 0.93, showing no significant differences across thrombin concentrations. In contrast, conventional confocal microscopy revealed an anomalous decrease in porosity with increasing fiber density. Additional structural parameters of the fibrin network—including fiber length, branching density, and average pore size—were assessed using confocal images acquired prior to sample expansion, as ExM provided a limited field of view with the same objective lens, representing a minor limitation of the technique.

The hydrogel expansion factor increased nonlinearly over time, reaching up to a 5-fold expansion. Imaging revealed a log-normal distribution of fibrin fiber thickness and a nonlinear, 2.22-fold decrease in mean fiber diameter as thrombin concentration increased from 0.3 to 1.5 U/mL. Measurements of average porosity using ExM images ranged from 0.92 to 0.93, showing no significant differences across thrombin concentrations. In contrast, conventional confocal microscopy revealed an anomalous decrease in porosity with increasing fiber density. Additional structural parameters of the fibrin network—including fiber length, branching density, and average pore size—were assessed using confocal images acquired prior to sample expansion, as ExM provided a limited field of view with the same objective lens, representing a minor limitation of the technique.

Acknowledgements and/or References (Optional):: This project is supported by the National Institute of Health grant (R21DE030294) and American Society of Hematology Junior Faculty Scholar Award.