Time-lapse microscopy of platelet α-granule movement during spreading. (A) Platelets were loaded with quantum dot 565 nanocrystals overnight and visualized during spreading on poly-L-lysine. Images were obtained every 30 seconds during spreading. Scale bar, 2.5 μm. (B) Images were taken immediately after adhesion (0 seconds) and at the indicated times thereafter. An overlay of a representative series of images demonstrates that the majority of the fluorescence remains localized to the central granulomere. However, movement of peripheral granules could be visualized. Peripheral granules imaged at different time points were pseudocolored to indicate position: 0 seconds (red), 30 seconds (yellow), 60 seconds (blue), and 90 seconds (purple). (C) Double fluorescence confocal microscopy of granules in spread platelets using anti–VAMP-7 antibody and endocytosed quantum dot 565 nanocrystals demonstrates peripheral colocalization of VAMP-7 with nanocrystals. (D) Double immunofluorescence staining of spread platelets demonstrates colocalization of P-selectin with quantum dot 565 nanocrystals in the platelet periphery. Scale bars, 5 μm.