Digital micrograph program

Resin-embedded blocks of stained pelleted platelets were first mounted on empty resin blocks for trimming in a Leica EM UC6 microtome. After the surface of the pellet was exposed, the block was remounted, exposed-side down, on an aluminum specimen pin (Gatan, Pleasanton, CA) using CircuitWorks Conductive Epoxy (CW2400), which grounded the sample electrically to the pin. Each sample was then trimmed again to expose the opposite side, and sputter-coated with a 40 nm gold layer. The trimmed block was imaged at an accelerating voltage of 1.8 kV in a Zeiss SIGMA-VP (variable pressure) scanning electron microscope (SEM) equipped with a Gatan 3View serial block face (SBF) imaging system [21 (link)–22 (link), 24 ]. The SEM was operated in the high vacuum mode with a 30 μm condenser aperture, and images containing 2000 × 2000 pixels were acquired using the Gatan DigitalMicrograph program with a pixel size of 5.5 nm in the x–y plane. The diamond knife of the SBF system was set to cut the block 1.2 mm/s with a slice thickness of 30 nm, thus providing a 30-nm pixel size along the z-axis. Resulting 250 image slices (total thickness of 7.5 μm) were assembled into a volume file and aligned, using the DigitalMicrograph software.