However, the effect of GSNO on fibrin clot architecture is unknown. To test the hypothesis that GSNO alters fibrin network microstructure, we used multiphoton microscopy to image human fibrin clots in their native state at 37uC and developed computer software to analyze clot structural elements. We found that over a four-fold concentration range GSNO shifted clot structure towards less dense coarse clots with thicker fibers, ultimately inhibiting clot formation. Additionally, at progressively higher GSNO concen- trations, first abnormal fibrin clusters developed within fibrin networks and ultimately fibrin networks were replaced by fibrin agglomerates. To the best of our knowledge, this is the first report of GSNO inducing fibrin particle formation. Taken together, GSNO altered human fibrin clot formation in such a way that network structures went beyond the bounds of the classically defined clot extremes of ‘‘fine’’ and ‘‘course’’ clots [14]. Methods Human plasma was obtained from the Canadian Blood Services blood bank for research purposes in accordance with research policies at the University of British Columbia. Data were analyzed anonymously. Plasma was collected from three healthy volunteers in acid citrate dextrose tubes and pooled as previously described [9,32]. All experiments were performed in triplicate. Fibrin Polymerization of Human Platelet-poor Plasma (PPP) Treated with S-nitrosoglutathione (GSNO) Platelet-poor plasma (PPP) was prepared by centrifuging plasma samples at 3000rpm for 20 minutes [1]. Aliquots were frozen at 280uC and thawed once on the day of each experiment. Fibrin polymerization reactions were carried out in 27 mL final volumes as follows: 21 mL PPP was mixed with 2 mL of S-nitrosoglu- tathione (GSNO, AG Scientific, Inc, San Diego, CA, final concentrations ranged from 10 mM–3.75 mM) or 2 mL PBS (phosphate buffered saline, ph 7.4, Sigma, St.Louis, MO) and incubated for 20 minutes in a closed tube placed in a 37uC water bath. The sample was then supplemented with 1 mL CaCl2 (Sigma, St.Louis, MO, final concentration 18 mM) and 1 mL Oregon Green labeled fibrinogen (5% vol/vol) to provide fluorescent imaging contrast. Lastly, the sample was rapidly mixed with 2 mL human thrombin (Chrono-log, Havertown, PA, final PPP concentration 1 U/ml). In control experiments, tissue plasminogen activator (tPA)/thrombin mixtures (ratios of 1/20 and 1/100) (Molecular Innovations Inc, Peary Court, MI) were used to verify that Oregon Green labeled fibrinogen did not interfere with normal clot formation and fibrinolysis. Once fibrin polymerization had been initiated, the PPP sample was quickly spotted on a cover slip pretreated with albumin to reduce surface effects and incubated (37uC) in the dark for 20 minutes. Since fibrin polymerization occurred rapidly under these conditions, we carefully applied the sample immediately after the onset of polymerization and let the fibrin clot develop in the standing platelet-poor plasma droplet. Samples reached their gel point in less than 5 minutes, with the exception of the highest GSNO concentration tested which failed to form a fibrin clot. Multiphoton Microscopy (MPM) Multiphoton microscopy is a relatively new imaging technology made possible by the development of femtosecond pulse lasers. In contrast to fluorescent confocal microscopy or spinning-disc microscopy where a single photon is used to excite a given fluorophore, in multiphoton microscopy (MPM), two photons of longer wavelength are used to simultaneously excite a sample fluorophore [33]. The advantage of MPM is that longer S-Nitrosoglutathione Alters Fibrin Architecture wavelengths allow for deeper sample penetration of both tissues [34] and fluids with less light scattering, less photobleaching and loss of fluorescent signal, as may occur with confocal microscopy. This occurs because multiphoton excitation is non-linear and limited to a small femtoliter focal volume at the focal point of the laser beam; whereas, one photon imaging creates a large cone of excitation within the sample. Nevertheless, multiphoton imaging can require high powers to excite imaging targets and may induce photobleaching. Accordingly, caution was taken in these experi- ments to limit sample exposure to laser energy in order to maintain fluorescent fiber structural integrity. Fibrin Clot Image Acquisition Using Multiphoton Microscopy Images were acquired twenty minutes after the onset of polymerization, and at least 15 minutes beyond the sample gel point. There was no evidence of fiber movement at the time of imaging indicating the clot network was stable. 900 nm fs pulses with a FWHM of 14 nm (Ti-Sapphire laser, Spectra Physics, Santa Clara, CA) were focused through a 636/1.2 water immersion objective mounted on an inverted Leica AOBS SP2 microscope system, (Leica Microsystems GmbH, Wetzlar, Ger- many). Fluorescent images (102461024 pixels, at 2 and 46 optical zoom, 8 bit image depth) were acquired using 400 Hz/3frame averages and an emission bandwidth of 500–650 nm. We imaged from the surface of the clot to a height of 100 microns and discovered that Nyquist sampling (,20 minute scan time) introduced sufficient energy to cause sample evaporation. To eliminate this problem and avoid introducing structural artifacts, yet still obtain images through the clot, we limited optical sectioning to 10 micron intervals to a height of 100 microns within the clot. Using this imaging protocol, we found no evidence of either sample evaporation or photobleaching. Image Processing and Quantitative Analysis of Human Fibrin Clot Structures Images of fibrin networks revealed complex structures that were difficult to analyze by visual inspection. To facilitate rapid quantitative analysis of fibrin clot structural elements, we developed computer software using the MATLAB computing environment (MathWorks, Natick, MA) which allowed us to visualize fibrin structures using intensity and binary images, contour maps and surface plots of fiber intensity. We anticipate that improved imaging methods will enhance quantification of native fibrin networks and will help our understanding of the relationships between endogenous and exogenous factors and fibrin clot architecture. All digital images were subjected to the same background subtraction, normalization and thresholding image processing steps. The starting point for network analysis was the identification of individual fibers and the determination of fibrin fiber density. This was accomplished by sampling images along equally spaced test lines, as shown in Figure S1. Since fiber orientation within a fibrin network is random, the sampling procedure is effectively a random sample of fibrin fibers. The software detected and numbered each fiber on the basis of its fluorescent signal, marked its location and stored its fluorescence intensity and image coordinates and mapped fiber position back into the original image. The computer program is interactive, allowing the user to inspect all identified fibers (Figure S1, panel C) and edit either false positives or false negatives. Since fiber diameter is directly related to lateral aggregation of fluorescently labeled fibrin monomers [9], the program next determined the local maximum fiber intensity and measured the fiber diameter at PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43660