Figure 2
Figure 2. Fluorescent fibrin fibers observed and characterized in TIRFM. (A) Typical TIRFM image of fluorescently labeled fibrin fibers formed by mixing thrombin into recalcified plasma and then insertion into the flow chamber. There was no preferential orientation of the fibers because of flow in the chamber, but most fibers were curved because of shear forces. (B) TIRFM observation chamber (2 clean glass coverslips on top of each other, separated laterally by double-stick tape to create a 75-μm thick chamber) allowed real-time observation of fibrin polymerization in TIRFM by introducing thrombin solution at one side of the chamber (green region) and allowing its diffusion into the region with fluorescent fibrinogen molecules (red region). (C) TIRFM images of the kinetics of growth of fibrin clot 1 molecule at a time. Growth of fibers continued for tens of minutes in the observation region, arbitrarily chosen in the center of the chamber close to the interface of thrombin and plasma. Frame 1 shows fluorescent fibrinogen in the central area of the chamber before introducing thrombin, and frames 2 and 3 were taken at 4.30 and 21 minutes after thrombin insertion. The sample was kept in the dark between frames 2 and 3 to avoid bleaching and thus show real lateral growth. (D-E) Graphs of fiber growth as a function of time, plotted as either number of molecules in the cross section of fiber, c, or hydrated fiber diameter, D. The 1-second time-point is the first moment of observation of the network after contact of thrombin with fibrinogen. According to the single-molecule calibration, lateral growth of fibers went up to thousands of molecules across (D), but some fiber regions reached an early stationary state (inset). Calculated diameters of the hydrated fibers (E) showed growth of up to 950 nm in 250 seconds for the largest fibers, and are in the same range as fiber diameters measured in unbleached fluorescence images, but more accurate. Regions of fibers analyzed were between branch points or intersections of fibers and had lengths from 2 to 10 μm. Image sequences were recorded at 1 frame/s. Resolution in all TIRFM images was 80 nm/pixel. Images presented are raw data with no bleaching correction performed for the frames in (C). Scale bar is 3 μm in panel A and 2.5 μm in panel C.

Fluorescent fibrin fibers observed and characterized in TIRFM. (A) Typical TIRFM image of fluorescently labeled fibrin fibers formed by mixing thrombin into recalcified plasma and then insertion into the flow chamber. There was no preferential orientation of the fibers because of flow in the chamber, but most fibers were curved because of shear forces. (B) TIRFM observation chamber (2 clean glass coverslips on top of each other, separated laterally by double-stick tape to create a 75-μm thick chamber) allowed real-time observation of fibrin polymerization in TIRFM by introducing thrombin solution at one side of the chamber (green region) and allowing its diffusion into the region with fluorescent fibrinogen molecules (red region). (C) TIRFM images of the kinetics of growth of fibrin clot 1 molecule at a time. Growth of fibers continued for tens of minutes in the observation region, arbitrarily chosen in the center of the chamber close to the interface of thrombin and plasma. Frame 1 shows fluorescent fibrinogen in the central area of the chamber before introducing thrombin, and frames 2 and 3 were taken at 4.30 and 21 minutes after thrombin insertion. The sample was kept in the dark between frames 2 and 3 to avoid bleaching and thus show real lateral growth. (D-E) Graphs of fiber growth as a function of time, plotted as either number of molecules in the cross section of fiber, c, or hydrated fiber diameter, D. The 1-second time-point is the first moment of observation of the network after contact of thrombin with fibrinogen. According to the single-molecule calibration, lateral growth of fibers went up to thousands of molecules across (D), but some fiber regions reached an early stationary state (inset). Calculated diameters of the hydrated fibers (E) showed growth of up to 950 nm in 250 seconds for the largest fibers, and are in the same range as fiber diameters measured in unbleached fluorescence images, but more accurate. Regions of fibers analyzed were between branch points or intersections of fibers and had lengths from 2 to 10 μm. Image sequences were recorded at 1 frame/s. Resolution in all TIRFM images was 80 nm/pixel. Images presented are raw data with no bleaching correction performed for the frames in (C). Scale bar is 3 μm in panel A and 2.5 μm in panel C.

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