Serial Block Face SEM Imaging of Platelets
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Corresponding Organization : University of Arkansas for Medical Sciences
Other organizations : National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health
Protocol cited in 1 other protocol
Variable analysis
- Trimming of the resin-embedded blocks of stained pelleted platelets
- Remounting the block with the exposed side down on an aluminum specimen pin
- Trimming the block again to expose the opposite side
- Sputter-coating the trimmed block with a 40 nm gold layer
- Imaging of the trimmed block in a Zeiss SIGMA-VP scanning electron microscope (SEM)
- Acquisition of images containing 2000 × 2000 pixels using the Gatan DigitalMicrograph program with a pixel size of 5.5 nm in the x–y plane
- Cutting of the block by the diamond knife of the SBF system at a rate of 1.2 mm/s with a slice thickness of 30 nm, providing a 30-nm pixel size along the z-axis
- Assembly of the resulting 250 image slices (total thickness of 7.5 μm) into a volume file and alignment using the DigitalMicrograph software
- Accelerating voltage of the SEM set at 1.8 kV
- SEM operated in the high vacuum mode with a 30 μm condenser aperture
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