Advertisement

Histochemistry and Cell Biology

, Volume 150, Issue 5, pp 545–556 | Cite as

STEM tomography of high-pressure frozen and freeze-substituted cells: a comparison of image stacks obtained at 200 kV or 300 kV

  • Paul Walther
  • Andrea Bauer
  • Nadia Wenske
  • Alberto Catanese
  • Débora Garrido
  • Marion Schneider
Original Paper
  • 167 Downloads

Abstract

Scanning transmission electron microscopic (STEM) tomography of high-pressure frozen, freeze-substituted semi-thin sections is one of multiple approaches for three-dimensional recording and visualization of electron microscopic samples. Compared to regular TEM tomography thicker sample sections can be investigated since chromatic aberration due to inelastic scattering is not a limit. The method is ideal to investigate subcellular compartments or organelles such as synapses, mitochondria, or microtubule arrangements. STEM tomography fills the gap between single-particle electron cryo-tomography, and methods that allow investigations of large volumes, such as serial block-face SEM and FIB-SEM. In this article, we discuss technical challenges of the approach and show some applications in cell biology. It is ideal to use a 300-kV electron microscope with a very small convergence angle of the primary beam (“parallel” beam). These instruments are expensive and tomography is rather time consuming, and therefore, access to such a high-end microscope might be difficult. In this article, we demonstrate examples of successful STEM tomography in biology using a more standard 200-kV microscope equipped with a field emission tip.

Keywords

STEM tomography Cell biology Synapse Electron microscopy High pressure freezing Freeze substitution 

Notes

Acknowledgements

We thank Giada Frascaroli for the M2 macrophage samples. We thank Renate Kunz for preparing the up to 1-µm-thick sections and mounting them on the copper grids and Reinhard Weih for keeping all our equipment alive. We thank Clarissa Read (formerly known as Clarissa Villinger) and Reinhard Rachel, Regensburg, for helpful discussions. We thank Ingo Daberkow from Tietz Systems GmbH for his abundance of patience at the phone, answering our amateurish questions, and for always getting our STEM system back to work. We finally thank David Mastronarde and his team for developing the IMOD software and making it freely available for everybody and for the counseling via the IMOD list.

Supplementary material

418_2018_1727_MOESM1_ESM.mov (32.6 mb)
Supplementary material 1 (MOV 33369 KB)
418_2018_1727_MOESM2_ESM.mov (43 mb)
Supplementary material 2 (MOV 43990 KB)
418_2018_1727_MOESM3_ESM.mov (180.3 mb)
Supplementary material 3 (MOV 184668 KB)

References

  1. Aoyama K, Takagi T, Hirase A, Miyazawa A (2008) STEM tomography for thick biological specimens. Ultramicroscopy 109:70–80CrossRefGoogle Scholar
  2. Bauer A, Subramanian N, Villinger C, Frascaroli G, Mertens T, Walther P (2016) Megapinocytosis: a novel endocytic pathway. Histochem Cell Biol 145:617–627CrossRefGoogle Scholar
  3. Bauer A, Frascaroli G, Walther P (2017) STEM tomography unveils lysosomal tubular networks in macrophages. In: Laue M (ed) Microscopy conference 2017 (MC 2017)—proceedings LS2.P005, RegensburgGoogle Scholar
  4. Baumeister W (2004) Mapping molecular landscapes inside cells. Biol Chem 385:865–872CrossRefGoogle Scholar
  5. Biskupek J, Leschner J, Walther P, Kaiser U (2010) Optimization of STEM tomography acquisition—a comparison of convergent beam and parallel beam STEM tomography. Ultramicroscopy 110:1231–1237CrossRefGoogle Scholar
  6. Catanese A, Garrido D, Walther P, Roselli F, Boeckers TM (2018) Nutrient limitation affects presynaptic structures through dissociable Bassoon autophagic degradation and impaired vesicle release. J Cereb Blood Flow Metab.  https://doi.org/10.1177/0271678X18786356 CrossRefPubMedGoogle Scholar
  7. Daems WT, Wisse E (1966) Shape and attachment of the cristae mitochondriales in mouse hepatic cell mitochondria. J Ultrastruct Res 16:123–140CrossRefGoogle Scholar
  8. Giehl K, Bachem M, Beil M, Böhm BO, Ellenrieder V, Fulda S, Gress TM, Holzmann K, Kestler HA, Kornmann M, Menke A, Möller P, Oswald F, Schmid RM, Schmidt V, Schirmbeck R, Seufferlein T, von Wichert G, Wagner M, Walther P, Wirth T, Adler G (2011) Inflammation, regeneration, and transformation in the pancreas: results of the Collaborative Research Center 518 (SFB 518) at the University of Ulm. Pancreas 40(4):489–502CrossRefGoogle Scholar
  9. Han S, Kollmer M, Markx D, Claus S, Walther P, Fändrich M (2017) Amyloid plaque structure and cell surface interactions of β-amyloid fibrils revealed by electron tomography. Sci Rep 7:43577CrossRefGoogle Scholar
  10. Hochapfel F, Denk L, Maaßen C, Zaytseva Y, Rachel R, Witzgall R, Krahn MP (2018) Electron microscopy of Drosophila garland cell nephrocytes: optimal preparation, immunostaining and STEM tomography. J Cell Biochem.  https://doi.org/10.1002/jcb.26702 CrossRefPubMedGoogle Scholar
  11. Hohmann-Marriott MF, Sousa AA, Azari AA, Glushakova S, Zhang G, Zimmerberg J, Leapman RD (2009) Nanoscale 3D cellular imaging by axial scanning transmission electron tomography. Nat Methods 6:729–731CrossRefGoogle Scholar
  12. Höhn K, Sailer M, Wang L, Lorenz L, Schneider EM, Walther P (2011) Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography. Histochem Cell Biol 135:1–9CrossRefGoogle Scholar
  13. Hoppe W, Gassmann J, Hunsmann N, Schramm HJ, Sturm M (1974) Three-dimensional reconstruction of individual negatively stained yeast fatty-acid synthetase molecules from tilt series in the electron microscope. Z Physiol Chem 355:1483–1487Google Scholar
  14. Klumperman J, Raposo G (2014) The complex ultrastructure of the endolysosomal system. Cold Spring Harb Perspect Biol 6:a016857CrossRefGoogle Scholar
  15. Knoll G, Brdiczka D (1983) Changes in freeze-fractured mitochondrial membranes correlated to their energetic state dynamic interactions of the boundary membranes. Biochim Biophys Acta 733:102–110CrossRefGoogle Scholar
  16. Kollmer M, Meinhardt K, Haupt C, Liberta F, Wulff M, Linder J, Handl L, Heinrich L, Loos C, Schmidt M, Syrovets T, Simmet T, Westermark P, Westermark GT, Horn U, Schmidt V, Walther P, Fändrich M (2016) Electron tomography reveals the fibril structure and lipid interactions in amyloid deposits. Proc Natl Acad Sci USA 113:5604–5609CrossRefGoogle Scholar
  17. Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76CrossRefGoogle Scholar
  18. Liu W, Naydenov B, Chakrabortty S, Wünsch B, Hübner K, Ritz S, Cölfen H, Barth H, Koynov K, Qi H, Leiter R, Reuter R, Wrachtrup J, Boldt F, Scheuer J, Kaiser U, Sison M, Lasser T, Tinnefeld P, Jelezko F, Walther P, Wu Y, Weil T (2016) Fluorescent nanodiamond-gold hybrid particles for multimodal optical and electron microscopy cellular imaging. Nano Lett 16:6236–6244CrossRefGoogle Scholar
  19. McBride EL, Rao A, Zhang G, Hoyne JD, Calco GN, Kuo BC, He Q, Prince AA, Pokrovskaya ID, Storrie B, Sousa AA, Aronova MA, Leapman RD (2018) Comparison of 3D cellular imaging techniques based on scanned electron probes: Serial block face SEM vs. axial bright-field STEM tomography. J Struct Biol 202:216–228CrossRefGoogle Scholar
  20. Müller SA, Engel A (2001) Structure and mass analysis by scanning transmission electron microscopy. Micron 32:21–31CrossRefGoogle Scholar
  21. Nafeey S, Martin I, Felder T, Walther P, Felder E (2016) Branching of keratin intermediate filaments. J Struct Biol 194:415–422CrossRefGoogle Scholar
  22. Sanders P, Walther P, Moepps M, Hinz M, Mostafa H, Schaefer P, Pala A, Wirtz CR, Georgieff M, Schneider EM (2018) Mitophagy-related cell death mediated by vacquinol-1 and TRPM7 blockade in glioblastoma IV. In: For “Glioma—contemporary diagnostic and therapeutic approaches”. InTechOpen (in press) Google Scholar
  23. Schauflinger M, Fischer D, Schreiber A, Chevillotte M, Walther P, Mertens T, von Einem J (2011) The tegument protein UL71 of human cytomegalovirus is involved in late envelopment and affects multivesicular bodies. J Virol 85: 3821–3832CrossRefGoogle Scholar
  24. Schauflinger M, Villinger C, Mertens T, Walther P, von Einem J (2013) Analysis of human cytomegalovirus secondary envelopment by advanced electron microscopy. Cell Microbiol 15:305–314CrossRefGoogle Scholar
  25. Villinger C, Gregorius H, Kranz Ch, Höhn K, Münzberg C, von Wichert G, Mizaikoff B, Wanner G, Walther P (2012) FIB/SEM-tomography with TEM-like resolution for 3D imaging of high pressure frozen cells. Histochem Cell Biol 138:549–556CrossRefGoogle Scholar
  26. Villinger C, Schauflinger M, Gregorius H, Kranz C, Höhn K, Nafeey S, Walther P (2014) Three-dimensional imaging of adherent cells using FIB/SEM and STEM. Methods Mol Biol 1117:617–638CrossRefGoogle Scholar
  27. Walther P, Ziegler A (2002) Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water. J Microsc 208:3–10CrossRefGoogle Scholar
  28. Walther P, Wang L, Ließem S, Frascaroli G (2010) Viral infection of cells in culture—approaches for electron microscopy. Methods Cell Biol 96:603–618CrossRefGoogle Scholar
  29. Wolf SG, Houben L, Elbaum M (2014) Cryo-scanning transmission electron tomography of vitrified cells. Nat Methods 11:423–428CrossRefGoogle Scholar
  30. Wolf SG, Mutsafi Y, Dadosh T, Ilani T, Lansky Z, Horowitz B, Rubin S, Elbaum M, Fass D (2017) 3D visualization of mitochondrial solid-phase calcium stores in whole cells. Elife 6:e29929CrossRefGoogle Scholar
  31. Yakushevska AE, Lebbink MN, Geerts WJ, Spek L, van Donselaar EG, Jansen KA, Humbel BM, Post JA, Verkleij AJ, Koster AJ (2007) STEM tomography in cell biology. J Struct Biol 159:381–391CrossRefGoogle Scholar
  32. Yin X, Ziegler A, Kelm K, Hoffmann R, Watermeyer P, Alexa P, Villinger C, Rupp U, Schlüter L, Reusch TBH, Griesshaber E, Walther P, Schmahl WW (2018) Formation and mosaicity of coccolith segment calcite of the marine algae Emiliania huxleyi. J Phycol 54:85–104CrossRefGoogle Scholar
  33. Zierold K, Steinbrecht A (1987) Cryofixation of diffusible elements in cells and tissues for electron probe microanalysis. In: Steinbrecht RA, Zierold K (eds) Cryotechniques in biological electron microscopy. Springer, Heidelberg, pp 3–34Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Paul Walther
    • 1
  • Andrea Bauer
    • 1
  • Nadia Wenske
    • 1
  • Alberto Catanese
    • 2
  • Débora Garrido
    • 2
  • Marion Schneider
    • 3
  1. 1.Central Facility for Electron MicroscopyUlm UniversityUlmGermany
  2. 2.Institute of Anatomy and Cell BiologyUlm UniversityUlmGermany
  3. 3.Division of Experimental AnesthesiologyUniversity Hospital UlmUlmGermany

Personalised recommendations