Type of presentation: Oral

ID-8-O-3291 STEM tomography of blood platelets in 1.5-µm thick sections

Aronova M. A.1, Sousa A. A.1, Pfeifer C. R.1, Shomorony A.1, He Q.1, Zhang G.1, Pokrovskaya I. D.2, MacDonald L.2, Prince A. A.2, Storrie B.2, Leapman R. D.1
1National Institute of Biomedical Imaging and Bioengineering, NIH, Bethesda, MD, USA, 2Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, USA
leapmanr@mail.nih.gov

Electron tomography in the scanning transmission electron microscope (STEM) can be performed on µm-thick plastic-embedded specimens without effects of chromatic aberration because there are no imaging lenses after the specimen [1-4]. 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 [2].

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 α-granules, which contain important proteins released when platelets are activated [5].

Electron tomograms were acquired using an FEI Tecnai TF30 (S)TEM equipped with a field-emission gun and operating at an acceleration voltage of 300 kV. The instrument was equipped with an axial bright-field STEM detector. Specimens were prepared by immediate paraformaldehyde fixation, followed by platelet purification, then either further chemical fixation with glutaraldehyde or high pressure freezing and freeze substitution with acetone/osmium tetroxide fixative, and finally dehydration, epon embedding, and U and Pb staining of the sections. Tomograms were reconstructed from dual-axis bright-field STEM tilt series using the IMOD program [6].

Although there is extensive overlap of structure in axial bright-field STEM images of 1.5-µm thick sections even at a tilt angle of 0° (Fig. 1A), it is possible to reconstruct blood platelets from dual-axis tilt series (Fig. 1B). Orthoslices through a tomographic reconstruction of cells oriented parallel to the section (Figs. 2A-C) and oriented perpendicular to the section (Figs. 3A-C) reveal ultrastructural features including the open canalicular system of membranes, mitochondria, and α-granules. The advantage of STEM tomography is that large structures within the platelets can be imaged in their entirety. Work is in progress to compare the ultrastructure of α-granules in resting and activated platelets.

[1] A.E. Yakushevska et al., J. Struct. Biol. 159 (2007) 381.

[2] M.F. Hohmann-Marriott, A.A. Sousa et al., Nature Methods 6 (2009) 729.

[3] A.A. Sousa et al., Ultramicroscopy 109 (2009) 213.

[4] A.A. Sousa et al., J. Struct. Biol. 174 (2011) 107.

[5] J. Kamykowski et al., Blood 118 (2011) 1370.

[6] J.R. Kremer, D.N. Mastronarde, J. Struct. Biol. 116 (1996) 71.

Research supported by the intramural program of NIBIB, NIH. Work in the Storrie laboratory was supported in part by NIH grant R01 HL119393.

Fig. 1: (A) STEM image of a 1.5-µm thick section of a platelet recorded at 0°-tilt showing superposition of structures; (B) dual-axis STEM tomographic reconstruction showing 3D volume.

Fig. 2: (A-C) Three orthoslices through STEM tomographic reconstruction of 1.5-µm thick section of human platelets showing cell oriented in plane of section: open canalicular system of membranes (ocs), mitochondria (m), α-granules (αg).

Fig. 3: (A-C) Three orthoslices through STEM tomographic reconstruction of 1.5-µm thick section of human platelets showing several cells oriented perpendicular to the plane of the section: open canalicular system of membranes (ocs), mitochondria (m), α-granules (αg).