Electron Tomography and Correlative Approaches in Platelet Studies

  • Kasia B. Engberts
  • Cor Seinen
  • Willie J. C. Geerts
  • Harry F. G. HeijnenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1812)


Blood platelets play a central role in the arrest of bleeding and the development of thrombosis. Unraveling the complex processes of platelet biogenesis from megakaryocytes, platelet adhesion, aggregation, and secretory responses are important topics in the field of hemostasis and thrombosis. Analysis of the ultrastructural changes that occur during these processes is essential for understanding the rapid membrane dynamics and has contributed substantially to our present knowledge of platelet formation and functioning. Recent developments in real-time imaging, correlative light and electron microscopy imaging (CLEM), and 3D (cryo) electron microscopy and tomography offer exciting opportunities to improve studies of the platelet adhesive responses and secretion at the ultrastructural level in a close to native environment. In this chapter we discuss and illustrate cryo preparation techniques (high-pressure freezing, vitrification), correlative LM and EM workflows, and 3D cryo-electron tomography that we apply in our current research projects.

Key words

TEM Immuno-EM Fixation and cryo-immobilization High pressure freezing EM tomography Cryo-EM Correlative light and electron microscopy (CLEM) 



Electron microscopy


Electron tomography


Correlative light and electron microscopy


Freeze substitution


High-pressure freezing


Liquid nitrogen


Charge coupled device




von Willebrand factor


Simultaneous iterative reconstruction technique


Missing Wedge

Missing information due to limited tilt angles


Manual drawing of contour lines in slices of a tomogram


Computed 3D volume reconstruction of a specimen by using multiple projection images

Supplementary material

Supplementary Movie 1

The movie shows the subsequent analytical steps for RT-CLEM and tomography of platelet whole mounts using the iCorrtm. Platelets spread conjugated anti-vWF and 10 nm Protein A gold. Regions of interest (green IF spots, reflecting luminal vWF release) were sequentially imaged in IF and TEM mode using the iCorr software package (iCorrtm FEI Company). Tilt series were recorded and aligned using the IMOD softwareon fibrinogen-coated EM supports were double immunolabeled with Alexa 488 (MP4 29686 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Kasia B. Engberts
    • 1
  • Cor Seinen
    • 2
  • Willie J. C. Geerts
    • 1
  • Harry F. G. Heijnen
    • 2
    • 3
    Email author
  1. 1.Bijvoet Center for Biomolecular ResearchUtrecht UniversityUtrechtThe Netherlands
  2. 2.Laboratory of Clinical Chemistry and HematologyUniversity Medical Center UtrechtUtrechtThe Netherlands
  3. 3.Department of Cell Biology, Cell Microscopy CoreUniversity Medical Center UtrechtUtrechtThe Netherlands

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