Schematic representation of the ₂-AP–based peptide that is bimodally labeled with rhodamine and a diethylene triamine pentaacetic acid-chelate (Bi-₂AP-CA) and the corresponding mass spectra. Matrix-assisted laser desorption/ionization mass spectrometry shows a molecular mass of 2,832.92 g/mol for the Bi-₂AP-CA. Substitution of only one amino acid (Q³A³) results in a bimodal control CA (Bi-con-CA).

Results of TPLSM and MRI for human (A to F) and murine (G to J) thrombi incubated with the bimodal ₂-AP–based contrast agent (Bi-₂AP-CA) (A, B, E to H) and the bimodal control CA (Bi-con-CA) (C, D, I to J). For TPLSM, red color originates from the rhodamine-labeled CAs. Green color indicates the fibrin network due to use of fibrinogen–Oregon green 488. Yellow color illustrates colocalization of red and green. Hyperintense MR signals were found at the edge of thrombi incubated with Bi-₂AP-CA (A, G). Lower MR signal intensities were found at the edge of a thrombus exposed to dansylcadaverine and Bi-₂AP-CA (E). MRI = magnetic resonance imaging; TPLSM = 2-photon laser-scanning microscopy.

(A) Factor XIII staining. (B) ₂-Antiplasmin staining. Positive staining is depicted in red/pink color. Factor XIII staining (scattered spots) was present throughout the whole thrombus while ₂-antiplasmin staining was mainly depicted at the edge of the thrombus (arrows). Bars = 50 µm.

Middle panels demonstrate an overview of thromboemboli, from top to bottom: hematoxylin and eosin stained, immunohistochemically stained for fibrin, factor XIII, and ₂-antiplasmin. Bars = 300 µm. Left panels show enlargements of boxed areas indicating an organized part of the thrombo-emboli. In the enlarged FXIII stained area clear cellular FXIII staining is visable. Bars = 50 µm. Right panels show enlargements of boxed areas indicating a fresh segment (arrows). Bars = 50 µm.

Relative fluorescence measured in blood samples decreases with time after contrast administration (A). Half-lives of the CAs are 14.1 ± 8.4 min and 16.3 ± 4.0 min for Bi-₂AP-CA and Bi-con-CA respectively. CAs are mainly cleared through the kidney (B and C). In B and C, red indicates bimodal CA in kidney tubuli. CA = constrast agent.

In vivo MRI (A to C) and ex vivo TPLSM results (E to G) of early thrombus formation and 24- to 48-h thrombi in murine carotid arteries after injection of the bimodal ₂-antiplasmin–based CA (Bi-₂AP-CA) (A, C, E, and G) and the bimodal control CA (Bi-con-CA) (B, F). Contrast-to-noise ratios (CNRs) are increased in the early phases of thrombus formation after Bi-₂AP-CA administration (D). In E to G, red indicates Bimodal CA, and green indicates nuclear stain. Lumen is located between dotted lines. Blue arrows indicate leukocytes trapped in the thrombus. Elongated cells are smooth muscle cells (orange arrows) and endothelial cells (red arrow). Abbreviations as in Figures 2 and 5.