Supplemental Methods
Fibrin clots were formed within a mylar bounded chamber around a 0.280 mm diameter cylindrical cast (Figure S1A),
later removed to permit the introduction of the microbubbles. The clinical microbubble contrast agent Definity (Lantheus
Medical Imaging) was employed. The clots were situated within a water tank positioned under a microscope (Olympus BXFM,
10x2 lens – UMPLFN10XW) equipped with a fast frame camera (Photron, APX-RS; 10kfps) (Figure S1B).
FIG. S1. A-Illustration of the cylindrical fluid/clot boundary (fluid in green and fibrin clot in blue). B-Side and top view of the channel
cast within the clot.
The optical field of view (440x440µm) was co-registered with the focal point of the transducer (Valpey Fisher, Diameter1”; f#-1; center frequency 1 MHz) using a glass bead (diameter ~200µm). The transducer beam was oriented perpendicular to the
clot channel, with its focus (and the optical field of view) situated at the center of the channel depth, opposite to the location of
the transducer (Figure S1a). 1 MHz pulsed US was applied in the form of 20 successive 1 ms bursts at a 15% duty cycle (5 ms
spacing). The transmit peak rarefactional pressures applied were 0.2, 0.4, 0.8, and 1.6 MPa which were measured with a needle
hydrophone (HPM04/1, Precision Acoustics). Immediately prior to commencing the acoustic exposures, a buffer solution (TrisHCl, pH 7.4) containing microbubbles (1:5000 dilution ratio) and 200 nm fluorescent beads (Invitrogen Fluospheres, carboxylate
modified, excitation/emission-540/560 nm; dilution ratio 1:500) was introduced into the channel, the latter being used as a
marker to assess the potential for channel fluid uptake into the clots. Once the exposures were completed, the clot chamber was
then removed and 3D two-photon microscopy (Olympus, FV 1000 MPE 25x lens – XLPlanN25XW) was employed to assess
damage to the fluorescently tagged fibrin network and for the presence of fluorescent beads. Volumetric data sets were created
comprised of a series of 2D images (640 μm horizontally) covering 0.2 mm in depth (2 μm spacing) and 1.6 mm laterally along
the channel. The presence of tunnels left by bubbles penetrating the fibrin network was assessed by visual inspection (Figure 3).
Differences in the number of tunnels between cases was assessed with a two-tailed unpaired Students t-test, with p<0.05
considered to be significant.
In order to capture the path of penetrating bubbles in a single image minimum intensity projections were performed in
ImageJ (http://rsb.info.nih.gov/ij/) treating the sequence of 2D fast frame images as a 3D volume. Segmentation of the boundary
was accomplished using the NeuronJS2 plugin in ImageJ. NeuronJ is a semi-automated routine developed to aid in the tracing and
quantification of filamentous image structures. The output of this program was a sequence of binary images in which non-zero
values corresponded to the identified boundary. The segmented images were then visualized in Matlab™ (Mathworks, Natick,
MA) in the form of mesh plots with displacement depicted on the z-axis. Average boundary displacement was arrived upon by
calculating the average boundary location for every frame and subtracting the average boundary location of the first frame. 3D
rendering of the two-photon tunnel images was accomplished using a commercial software package (Imaris, Bitplane).
Detailed materials preparation
Following the work of Francis et al.S1fibrin clots were formed by adding thrombin (Sigma-Aldrich, 1NIH U/ml, final
concentration) to a solution containing human FXIII and fibrinogen at final concentrations of 20 LU/ml and 3 mg/ml,
respectively. The fibrinogen component was comprised of fluorescently tagged fibrinogen (Invitrogen, AlexaFluor-488) and
native fibrinogen (Hyphen-BioMed)at a concentration ratio of 1:10. The purified native fibrinogen and fluorescent fibrinogen
sources were reconstituted in Tris-HCl buffer at room temperature and placed on a slow rocker overnight to ensure the
lyophilized protein completely dissolved. The buffer was comprised of 50mM Tris-base, 100mM NaCl, brought to a pH of 7.4
with HCl and filtered with 0.2µm PVDF syringe filters (Millipore). This buffer was also used as the perfusion buffer in which the
Definity microbubbles were diluted. The dissolved native fibrinogen, fluorescent fibrinogen and FXIII were mixed (final
concentrations of 2.73mg/ml, 0.27mg/ml, and 20 LU/ml, respectively), aliquoted into 1ml amounts, and stored at -80oC. A stock
thrombin solution was also prepared in Tris-HCl buffer which was aliquoted into 0.2ml amounts and stored at -20oC; it contained
thrombin (Sigma-Aldrich) and CaCl2 which after mixing with the fibrinogen solution were present at final concentrations of
1NIH U/ml and 20 mM, respectively. 18 hours before the experiment the fibrinogen solution was thawed for 1 hour at room
temperature and placed in a fridge at 4oC overnight. On the morning of the experiment, prior to clot formation, the fibrinogen and
thrombin solutions were degassed. Clot formation was initiated by the addition of 20 µl of the thrombin solution. The clotting
solution was immediately injected into the chamber surrounding a cylindrical cast of polyethylene tubing (Advanced Polymers,
~280µm outer diameter) which was primed with Tris-HCl buffer. Injection occurred over 1 minute and the clotting time was
approximately 10 minutes (assessed by eye when opacity of the solution ceased to increase). Clot formation was allowed to come
to completion over 1 hour at room temperature at which point the cylindrical cast was removed and replaced with gas
equilibrated Tris-HCl buffer. The closed chamber was placed under the microscope in a tank of degassed water which was kept at
37oC with a temperature regulated water circulator (Lauda). All sonications were performed 2 hours after clot formation.
References
S1
J. V Braaten, C.W. Francis, and R.A. Goss, J. Thromb. Haemost. 78, 1063 (1997).
E. Meijering, M. Jacob, J.-C.F. Sarria, P. Steiner, H. Hirling, and M. Unser, Cytometry. Part A : the Journal of the
International Society for Analytical Cytology 58, 167 (2004).
S2