TIRF Microscopy-Based Visualization of Phagosome Formation and Closure

Take a polymer-coated glass bottom dish containing IgG-opsonized red blood cells or RBCs adhered to its surface.

Place the dish on a total internal reflection fluorescence microscope stage, maintaining it at physiological temperature.

Add macrophages containing fluorescently labeled cytoplasmic peptides that bind to polymerized actin, facilitating cytoskeleton visualization.

During imaging, direct a laser beam at an angle superior to the critical angle, causing internal reflection and evanescent wave generation at the contact point.

The evanescent waves selectively illuminate the fluorophores at the glass-sample interface, allowing real-time visualization of phagocytosis.

Macrophage Fc receptors bind to the IgG-opsonized RBC, causing receptor clustering around the contact area.

Clustering triggers intracellular signaling pathways and GTPase activation, driving actin polymerization and dynamin recruitment, forming pseudopods.

The pseudopods extend around the RBC, forming a phagocytic cup.

Eventually, the pseudopod tips fuse. The accumulated dynamin mediates phagosome separation from the cell membrane, internalizing the opsonized RBC.