Scanning Transmission Electron Tomography of Blood Platelets in Thick Sections

Abstract

Electron tomography in the scanning transmission electron microscope (STEM) can be performed on sections of stained plastic-embedded tissues or cells of 1 to 2 micrometer thickness without effects of chromatic aberration because there are no imaging lenses after the specimen. By using a small STEM probe convergence angle of 1-2 mrad the geometrical broadening of the probe is restricted, which enables a spatial resolution of a few nanometers. Furthermore, by using an axial bright-field detector instead of the standard high-angle annular dark-field detector, image blurring due to multiple elastic scattering can be reduced in the lower part of the specimen. Here, we have applied STEM tomography to elucidate the 3D ultrastructure of human blood platelets, which are small anucleate blood cells that aggregate to seal leaks at sites of vascular injury and are important in the pathology of atherosclerosis and other diseases. Of particular interest are the morphological changes that occur in alpha-granules, which contain important proteins released when platelets are activated. Axial bright-field STEM electron tomographic tilt series were acquired at an accelerating voltage of 300 kV from 1.5-micrometer thick sections of platelets that had been prepared by rapid freezing and freeze-substitution; and the tomograms were reconstructed from dual-axis tilt series. The tomographic reconstructions revealed changes in ultrastructure that occurred on platelet activation including release of alpha granules through channels connecting to the plasma membrane. The research was supported by the intramural program of the National Institute of Biomedical Imaging and Bioengineering, and the research in the Storrie laboratory was supported in part by NIH grant R01 HL119393.