Measuring Fibrin Fiber Dimensions

All fibrin fibers were imaged in buffer (same buffers as described above under fibrin fiber preparation), using a combined atomic force microscope (AFM) (Topometrix Explorer, Veeco Instruments, Woodbury, NY) and inverted fluorescence microscope (Axiovert 200 or Observer D, Zeiss, Göttingen, Germany). The fiber sample was placed between the AFM and optical microscope using a customized stage which allows the sample to be moved independently of either microscope [18 (link)–22 (link)]. Fibers were imaged in tapping mode with any one of the three cantilevers of AFM probe CSC-38 (MikroMasch, Wilsonville, OR) or an equivalent AFM probe (similar k and f). The spring constant, k, ranged from 0.03 N/m to 0.09 N/m, and the resonance frequency, f, ranged from 10 kHz to 20 kHz. Fibers were typically imaged at a 50% set point (50% of maximum free amplitude); the set point was adjusted so that the probe exerted the smallest possible normal force on the sample, while still making good contact with the surface. Feedback gains were adjusted as high as possible, without causing ringing. The fiber diameter, D, was determined by AFM imaging the fiber either on top of the ridge adjacent to where the fiber was manipulated (for the force measurements) or on the glass slide (for the fluorescence intensity measurements). The fiber cross-section was calculated assuming a cylindrical cross-section.