FIB-SEM of salivary gland secretory granules and measurements of granule diameter, actin thickness, and F-actin lattice diameter. WT mice were injected with 0.01 mg/kg ISOP and after 10 min the glands were processed for FIB-SEM and imaged. (A) Volume rendering of a segment of the APM (white) and the fused secretory granules (green). Bar, 1 µm. The inset shows a small vesicle of undetermined origin. Bar, 200 nm. (B) Example of secretory granules: non-fused (left) and at two different stages of integration (center and right). Upper panels—EM micrographs. Center panel—Volume rendering. Lower panels—Heat map of local curvature (red positive, blue negative). (C) Analysis of roundness and mean curvature of fused secretory granules. N = 19 granules, 2 animals. (D) Distribution of the granule diameters measured by FIB-SEM (N = 19 granules, 2 animals) or IVM (N = 55 granules, 25 cells, 5 animals). (E and F) Z-stacks of granules labeled with either the mTom probe (magenta) or Lifeact (green) were acquired by spinning disk microscopy. Single optical slices encompassing the largest granule cross section were selected. Using the line scan function in Fiji, a line was drawn across the center of the granules. (E) The fluorescence intensity plot generated exhibits two Gaussian intensity profiles. The peak-to-peak value (d) was used as measure of the diameter F. The diameters of the F-actin lattice were calculated using a similar strategy. However, during the thickening the diameters of the lattice were calculated as the distance D between the sides of the peaks at the half maximum intensity. The actin thickness was calculated as the average of the half maximum widths of the peaks (t1 and t2).