Super-resolution structured illumination and immunogold electron microscopy reveals distinct α-granule subpopulations in mouse proplatelets and human platelets. Mouse fetal liver derived MKs were isolated with a BSA gradient. Released mouse proplatelets were stained with antibodies against bFGF, ENDO, TSP, and VEGF and imaged with structured illumination microscopy. (A) A single slice of a composite stack of a released proplatelet. Scale bar is 10 µm. Magnified images show proplatelet shaft (1) and tip (2). (B) Distribution of α-granule cargo proteins were determined by overlaying different staining combinations. Scale bar is 1 µm. (C) Full image of proplatelet tip (left). White boxes highlight granules of distinct protein cargo, magnified on right. Scale bar is 1 µm. (D) Human platelets were probed with antibodies against bFGF, ENDO, TSP, and VEGF and imaged with structured illumination microscopy. Images are maximum intensity projections. Scale bar is 2 µm. (E) Double immunogold labeling of human platelet sections using anti-ENDO and antifibrinogen primary antibodies followed by staining with protein A–gold conjugated to 10 nm (fibrinogen; red arrows) or 15 nm (ENDO; white arrows). Scale bar is 100 nm.