S-Nitrosoglutathione Alters Fibrin Architecture Figure 4. GSNO reduces fibrin branchpoint density, but does not alter the fiber density/branchpoint ratio. Images of human fibrin clots in their native state were acquired using multiphoton microscopy and analyzed using custom designed computer software, as described in methods. Panels A,B,C show a multiphoton image with three branch points (B1,B2,B3) and their corresponding contour map and surface plots, respectively. Branch points are readily identifiable in surface plots, where three fibers intersect at the branch junction and the width of one fiber is approximately equal to the sum of the other fibers (F1 = F2+F3). As GSNO concentration increased, branch junctions per area decreased, panel D. However, the ratio of fiber density to branch point density was insensitive to GSNO concentration, panel E. *p,0.05, compared to control. Area is 59659 um2. Colour bars are image intensity. Scale bar is 4.25 um. doi:10.1371/journal.pone.0043660.g004 where the width of one fiber equals the sum of widths of the other two fibers (F1 = F2+F3) [6,7,37]. Fiber branching gives rise to the complex nature of fibrin networks. In Figure 4 we show how trifunctional junctions can be readily identified in multiphoton images (Figure 4A) using a combination of contour maps (Figure 4B) and surface plots (Figure 4C) of the region of interest. As GSNO increased, branch point density per unit area was found to decrease from 48.768.3 to zero (branch points/area), Figure 4D. However, when compared to changes in fiber density (Figure 2D), the ratio of fiber density to branch point density was found to be insensitive to GSNO concentration. Our finding is similar to the report that the fiber density/branch point density ratio is a constant, and generally independent of clotting conditions [8]. (Note, we list branch point (trifunctional junctions) statistics as xbranches in clot ‘‘read out’’ images, Figure 2, panels A,B,C,G.) PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43660