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Electron Tomography and Correlative Approaches in Platelet Studies

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

Abstract

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) 

Abbreviations

EM

Electron microscopy

ET

Electron tomography

CLEM

Correlative light and electron microscopy

FS

Freeze substitution

HPF

High-pressure freezing

LN2

Liquid nitrogen

CCD

Charge coupled device

3D

Three-dimensional

vWF

von Willebrand factor

SIRT

Simultaneous iterative reconstruction technique

Terminology

Missing Wedge

Missing information due to limited tilt angles

Contouring

Manual drawing of contour lines in slices of a tomogram

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)

References

  1. 1.
    Heijnen HFG, Korporaal SJA (2017) Platelet morphology and ultrastructure. In: Gresele P et al (eds) Platelets in thrombotic and non-thrombotic disorders, pathophysiology, pharmacology and therapeutics. Springer International Publishing AG, Basel, pp 21–37CrossRefGoogle Scholar
  2. 2.
    Behnke O (1968) An electron microscope study of the megacaryocyte of the rat bone marrow. I. The development of the demarcation membrane system and the platelet surface coat. J Ultrastruct Res 24(5):412–433CrossRefPubMedGoogle Scholar
  3. 3.
    Behnke O (1968) Electron microscopical observations on the surface coating of human blood platelets. J Ultrastruct Res 24(1–2):51–69CrossRefPubMedGoogle Scholar
  4. 4.
    Behnke O (1970) The morphology of blood platelet membrane systems. Ser Haematol 3(4):3–16PubMedGoogle Scholar
  5. 5.
    Behnke O, Forer A (1998) From megakaryocytes to platelets: platelet morphogenesis takes place in the bloodstream. Eur J Haematol Suppl 61:3–23PubMedGoogle Scholar
  6. 6.
    Morgenstern E (1982) Coated membranes in blood platelets. Eur J Cell Biol 26(2):315PubMedGoogle Scholar
  7. 7.
    Bentfeld-Barker ME, Bainton DF (1982) Identification of primary lysosomes in human megakaryocytes and platelets. Blood 59(3):472–481PubMedGoogle Scholar
  8. 8.
    Stenberg PE, Shuman MA, Levine SP, Bainton DF (1984) Redistribution of alpha-granules and their contents in thrombin-stimulated platelets. J Cell Biol 98(2):748–760CrossRefPubMedGoogle Scholar
  9. 9.
    White JG (1968) The substructure of human platelet microtubules. Blood 32(4):638–648PubMedGoogle Scholar
  10. 10.
    White JG (1968) Fine structural alterations induced in platelets by adenosine diphosphate. Blood 31(5):604–622PubMedGoogle Scholar
  11. 11.
    White JG, Clawson CC (1980) The surface-connected canalicular system of blood platelets–a fenestrated membrane system. Am J Pathol 101(2):353–364PubMedPubMedCentralGoogle Scholar
  12. 12.
    White JG (1998) Use of the electron microscope for diagnosis of platelet disorders. Semin Thromb Hemost 24(2):163–168CrossRefPubMedGoogle Scholar
  13. 13.
    Morgenstern E (1991) Aldehyde fixation causes membrane vesiculation during platelet exocytosis: a freeze-substitution study. Scanning Microsc Suppl 5(4):S109–S115PubMedGoogle Scholar
  14. 14.
    Murk JLAN, Posthuma G, Koster AJ, Geuze HJ, Verkleij AJ, Kleijmeer MJ, Humbel BM (2003) Influence of aldehyde fixation on the morphology of endosomes and lysosomes: quantitative analysis and electron tomography. J Microsc 212(1):81–90CrossRefPubMedGoogle Scholar
  15. 15.
    Posthuma G, Slot JW, Geuze HJ (1987) Usefulness of the immunogold technique in quantitation of a soluble protein in ultra-thin sections. J Histochem Cytochem 35(4):405–410CrossRefPubMedGoogle Scholar
  16. 16.
    Tokuyasu KT, Singer SJ (1976) Improved procedures for immunoferritin labeling of ultrathin frozen sections. J Cell Biol 71(3):894–906CrossRefPubMedGoogle Scholar
  17. 17.
    Liou W, Geuze HJ, Slot JW (1996) Improving structural integrity of cryosections for immunogold labeling. Histochem Cell Biol 106(1):41–58CrossRefPubMedGoogle Scholar
  18. 18.
    Slot JW, Geuze HJ (2007) Cryosectioning and immunolabeling. Nat Protoc 2(10):2480CrossRefPubMedGoogle Scholar
  19. 19.
    Riehle U, Hochli M (1973) In: Benedetti EL, Favard P (eds) In freeze-etching technique and applications. Society Francaise de Microscopy Electronique, Paris, pp 31–66Google Scholar
  20. 20.
    Studer D, Graber W, Al-Amoudi A, Eggli P (2001) A new approach for cryofixation by high-pressure freezing. J Microsc 203(3):285–294CrossRefPubMedGoogle Scholar
  21. 21.
    Vanhecke D, Graber W, Studer D (2008) Chapter 9 close-to-native ultrastructural preservation by high pressure freezing. Methods Cell Biol 88:151–164CrossRefPubMedGoogle Scholar
  22. 22.
    Valentijn KM, Valentijn JA, Jansen KA, Koster AJ (2008) A new look at Weibel-Palade body structure in endothelial cells using electron tomography. J Struct Biol 161(3):447CrossRefPubMedGoogle Scholar
  23. 23.
    Berriman JA, Li S, Hewlett LJ, Wasilewski S, Kiskin FN, Carter T, Hannah MJ, Rosenthal PB (2009) Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells. Proc Natl Acad Sci 106(41):17407–17412CrossRefPubMedGoogle Scholar
  24. 24.
    van Nispen tot Pannerden H, de Haas F, Geerts W, Posthuma G, van Dijk S, Heijnen HF (2010) The platelet interior revisited: electron tomography reveals tubular alpha-granule subtypes. Blood 116(7):1147–1156CrossRefPubMedGoogle Scholar
  25. 25.
    Valentijn KM, van Driel LF, Mourik MJ, Hendriks GJ, Arends TJ, Koster AJ, Valentijn JA (2010) Multigranular exocytosis of Weibel –Palade bodies in vascular endothelial cells. Blood 116(10):1807–1816CrossRefPubMedGoogle Scholar
  26. 26.
    Mourik MJ, Faas FGA, Valentijn KM, Valentijn JA, Eikenboom JC, Koster AJ (2014) Correlative light microscopy and electron tomography to study van Willebrand factor exocytosis from vascular endothelial cells. Methods Cell Biol 124:71–92CrossRefPubMedGoogle Scholar
  27. 27.
    Agronskaia AV, Valentijn JA, van Driel LF, Schneijdenberg CTWM, Humbel BM, van Bergen en Henegouwen PMP, Verkleij AJ, Koster AJ, Gerritsen HC (2008) Integrated fluorescence and transmission electron microscopy. J Struct Biol 164(2):183–189CrossRefPubMedGoogle Scholar
  28. 28.
    Faas FGA, Bárcena M, Agronskaia AV, Gerritsen HC, Moscicka KB, Diebolder CA, van Driel LF, Limpens RWAL, Bos E, Ravelli RBG, Koning RI, Koster AJ (2013) Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy. J Struct Biol 181(3):283–290CrossRefPubMedGoogle Scholar
  29. 29.
    de Boer P, Hoogenboom JP, Giepmans BNG (2015) Correlated light and electron microscopy: ultrastructure lights up! Nat Methods 12(6):503–513CrossRefPubMedGoogle Scholar
  30. 30.
    Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1):71CrossRefPubMedGoogle Scholar
  31. 31.
    van Donselaar EG, Posthuma G, Zeuschner D, Humbel BM, Slot JW (2007) Immunogold labeling of cryosections from high-pressure frozen cells. Traffic 8(5):471–485CrossRefPubMedGoogle Scholar
  32. 32.
    Al-Amoudi A, Chang JJ, Leforestier A, McDowall A, Salamin LM, Norlén LP, Richter K, Blanc NS, Studer D, Dubochet J (2004) Cryo-electron microscopy of vitreous sections. EMBO J 23(18):3583–3588CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Schaffer M, Engel BD, Laugks T, Mahamid J, Plitzko JM, Baumeister W (2015) Cryo-focused ion beam sample preparation for imaging vitreous cells by cryo-electron tomography. Bio Protoc 5(17):e1575CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Heijnen HF, Oorschot V, Sixma JJ, Slot JW, James DE (1997) Thrombin stimulates glucose transport in human platelets via the translocation of the glucose transporter GLUT-3 from alpha-granules to the cell surface. J Cell Biol 138(2):323–330CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Heijnen HF, Debili N, Vainchencker W, Breton-Gorius J, Geuze HJ, Sixma JJ (1998) Multivesicular bodies are an intermediate stage in the formation of platelet alpha-granules. Blood 91(7):2313–2325PubMedGoogle Scholar
  36. 36.
    Hoppe W (1974) Towards three-dimensional "electron microscopy" at atomic resolution. Naturwissenschaften 61(6):239–249CrossRefPubMedGoogle Scholar
  37. 37.
    Baumeister W, Grimm R, Walz J (1999) Electron tomography of molecules and cells. Trends Cell Biol 9(2):81–85CrossRefPubMedGoogle Scholar
  38. 38.
    Diebolder CA, Koster AJ, Koning RI (2012) Pushing the resolution limits in cryo electron tomography of biological structures. J Microsc 248(1):1–5CrossRefPubMedGoogle Scholar
  39. 39.
    Gilbert PFC (1972) The reconstruction of a three-dimensional structure from projections and its application to electron microscopy. II direct methods. Proc R Soc London B Ser Biol Sci 182:89–102CrossRefGoogle Scholar
  40. 40.
    Kühlbrandt W (2014) The resolution revolution. Science 343(6178):1443–1444CrossRefPubMedGoogle Scholar
  41. 41.
    Karreman MA, van Donselaar EG, Gerritsen HC, Verrips CT, Verkleij AJ (2011) VIS2FIX: a high-speed fixation method for immuno-electron microscopy. Traffic 12(7):806–814CrossRefPubMedGoogle Scholar

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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