Live cell fluorescence and super-resolution structured illumination microscopy reveal distinct subpopulations of α-granules and VAMPs, in mature mouse MKs. Mouse fetal liver derived MKs were cultured overnight with Alexa 488-fibrinogen (FG488) and Alexa 568-endostatin (ENDO568) or Alexa 488-fibrinogen and Alexa 546-fibrinogen (FG546). (A) Structured illumination microscopy of MKs containing fluorescently tagged ENDO and fibrinogen. The yellow boxes, and their corresponding magnified insets, highlight separation of FG488/ENDO568 in the upper panels and colocalization of FG488/FG546 in the lower panels. Images are maximum intensity projections. For FG488/ENDO568, scale bar is 5 µm for full images, and 2 µm for the inset. For FG488/FG546, scale bar is 10 µm for full images, and 5 µm for inset. (B) Time lapse imaging of a proplatelet containing FG488 and ENDO568. White arrowhead denotes fibrinogen-containing granule; open arrowhead denotes ENDO-containing granule. Images were taken every 5 minutes for 1 hour. Scale bar is 5 µm. (C) Structured illumination microscopy of VAMP-3 (green) and VAMP-8 (red). Note that the proplatelet shown in the insets are circular in structure. Images are maximum intensity projections. Scale bar is 25 µm for full images, and 8 µm for insets. For VAMP-3 and VAMP-8 costaining, the Pearson’s R value is 0.21 ± 0.02. Three proplatelet producing MKs were analyzed.