In vivo real-time imaging during cortical photothrombotic occlusion in full-field photothrombosis.

Dual-color imaging was performed after tail-vein injection of a mixture of Rose Bengal with Rhodamine-B dextran and DyLight 488-conjugated activated platelet antibody in all mice except for sham mice, which were replaced with normal saline instead of Rose Bengal. The fluorescence images were collected in both the 520~550 nm (green pseudo-color) and 573~631 nm (red pseudo-color) channels. The green pseudo-color represents activated platelets that form thrombi generated by photochemical stimulation inside blood vessels, and the red pseudo-color represents plasma flowing through the blood vessels. The direction of blood flow is indicated by a yellow arrow and is not indicated from the point when a blood clot blocks the blood vessel. The blue dotted circles indicate the 532-nm laser pattern used for each experimental group. The diameter of each vessel visible in the overall image is indicated by a white number and was measured from the corresponding white dotted line. Full-field photothrombosis was performed using a 320-μm diameter laser circular pattern. The 320-μm pattern covers the entire field of view in the image. Experiment was stopped when it was determined that the blood clot was sufficiently hardened in the blood vessel to block blood flow. All experiments in this study were limited to 10 minutes and experimentally obtained time was judged sufficient to complete occlusion after thrombus formation under our experimental conditions. Scale bar, 50 μm