Volume scanning electron microscopy reveals tight associations between forming WPBs and the Golgi apparatus in native HUVECs. The Golgi area was examined and imaged using focused ion beam scanning electron microscopy (FIB-SEM). (A) Three-dimensional model of the total imaged volume: 11.4 × 8.5 × 16 µm. The Golgi apparatus is shown in green; the WPBs associating with the Golgi are in red; peripheral WPBs are in orange; nucleus is in dark blue; and the cell membrane is shown in light blue. (B) FIB-SEM slice showing the Golgi, an associating immature WPB (*), and mature WPBs (**). Note that the mature WPB is much brighter due to its concentrated protein compared with the immature WPBs. The nucleus (Nu), the endoplasmic reticulum (Er), and the mitochondria (Mi) are also visible. (C-Cii) Series of slices from the imaged volume to show the tight association between the Golgi and an immature WPB. The data are shown in the x-y orientation. (C-Ci) Connection between the immature WPB and the Golgi (arrow). (Cii) A few VWF tubules (arrowheads) in the lumen of the immature WPB. (D-Dii) Series of slices from the imaged volume to show multiple associations between the Golgi and a single immature WPB. The data are shown in the x-z orientation. (D) Immature WPB in close proximity with the Golgi. (Di) Connection between the top part of the WPB and the Golgi. (Dii) Additional connections with the Golgi along the membrane of the WPBs, which were found at the bottom of the WPBs. See also supplemental Video 3. Scale bars: (A) 1 µm; (C-D) 500 nm. Volume-SEM data were acquired using an Auriga CrossBeam (Carl Zeiss Microscopy GmbH) SEM at an acceleration voltage of 1.7 kV and a beam current of 220 pA. Images were acquired using both the InLens secondary electron detector and the energy selected backscattered electron detector. Data were collected at ×25 000 magnification. Segmentation was performed in Amira (Visualization Sciences Group, FEI). Go, Golgi.