Intravital Fluorescence Microscopy to Study Microvascular Thrombus Formation

Intravital fluorescence microscopy is useful for studying the phototoxic induction of the formation of a thrombus — a blood clot — at a microvascular injury site.

Begin by taking an anesthetized hairless mouse injected with a phototoxic fluorophore-labeled polysaccharide. Place the mouse in a prone position on a heating platform to maintain the physiological temperature for proper blood circulation.

Place the apical part of the ear under an intravital fluorescence microscope. Upon excitation with light, the fluorophore emits fluorescence — allowing monitoring of the microvascular blood flow. Increase the light intensity to induce thrombus formation.

The fluorophore absorbs the high-intensity light and reacts with molecular oxygen to generate reactive oxygen species. These molecules cause oxidative stress in the endothelial cells, thus leading to cell damage and the exposure of the subendothelial matrix.

The damaged cells release thrombotic mediators that bridge the exposed collagen in the subendothelial matrix and the circulating platelets, which bind to the mediators via specific receptors.

The binding activates the platelets, causing the release of effector molecules that activate and recruit other circulating platelets to the injury site. Circulating fibrinogen proteins bind to the fibrinogen receptors on the activated platelets — linking them to form a platelet plug.

Visualize the restricted blood flow to confirm the platelet plug — the primary step of thrombus formation.