Transforming FIB-SEM Systems for Large-Volume Connectomics and Cell Biology

  • C. Shan XuEmail author
  • Song Pang
  • Kenneth J. Hayworth
  • Harald F. Hess
Part of the Neuromethods book series (NM, volume 155)


Isotropic high-resolution imaging of large volumes provides unprecedented opportunities to advance connectomics and cell biology research. Conventional focused ion beam scanning electron microscopy (FIB-SEM) offers unique benefits such as high resolution (<10 nm in x, y, and z), robust image alignment, and minimal artifacts for superior tracing of neurites. However, its prevailing deficiencies in imaging speed and duration cap the maximum possible image volume. We have developed technologies to overcome these limitations, thereby expanding the image volume of FIB-SEM by more than four orders of magnitude from 103 μm3 to 3 × 107 μm3 while maintaining an isotropic resolution of 8 × 8 × 8 nm3 voxels. These expanded volumes are now large enough to support connectomic studies, in which the superior z resolution enables automated tracing of fine neurites and reduces the time-consuming human proofreading effort. Moreover, by trading off imaging speed, the system can readily be operated at even higher resolutions achieving voxel sizes of 4 × 4 × 4 nm3, thereby generating ground truth of the smallest organelles for machine learning in connectomics and providing important insights into cell biology. Primarily limited by time, the maximum volume can be greatly extended.

In this chapter, we provide a detailed description of the enhanced FIB-SEM technology, which has transformed the conventional FIB-SEM from a laboratory tool that is unreliable for more than a few days to a robust imaging platform with long-term reliability: capable of years of continuous imaging without defects in the final image stack. An in-depth description of the systematic approach to optimize operating parameters based on resolution requirements and electron dose boundary conditions is also explicitly disclosed. We further explore how this technology unleashes the full potential of FIB-SEM systems, revolutionizing volume electron microscopy (EM) imaging for biology by gaining access to large sample volumes with single-digit nanoscale isotropic resolution.

Key words

Focused ion beam scanning electron microscopy (FIB-SEM) Volume electron microscopy 3D imaging Large volume 3D structure Isotropic resolution Connectomics Cell biology Drosophila Mouse brain Mammalian cell 



We would like to thank David Peale and Patrick Lee for consulting support in system modification. We also thank Zhiyuan Lu, Gleb Shtengel, David Hoffman, Amalia H. Pasolli, Kathy Schaefer, Aubrey Weigel, Nadine Randel, Michael J. Winding, and Graham Knott for EM sample preparation. We gratefully acknowledge Patrick Naulleau, Ian A. Meinertzhagen, and Steve Plaza for reviewing the manuscript and providing timely feedback. Our gratitude extends to Janelia FlyEM connectome program, in particular Gerry Rubin and Steve Plaza for their leadership. We were solely funded by the Howard Hughes Medical Institute.


  1. 1.
    Xu CS, Hayworth KJ, Lu Z et al (2017) Enhanced FIB-SEM systems for large-volume 3D imaging. eLife 6:e25916. Scholar
  2. 2.
    Xu, CS, Hayworth KJ, Hess HF (2020) Enhanced FIB-SEM systems for large-volume 3D imaging. US Patent 10,600,615, 24 Mar 2020Google Scholar
  3. 3.
    Heymann JA, Hayles M, Gestmann I et al (2006) Site-specific 3D imaging of cells and tissues with a dual beam microscope. J Struct Biol 155:63–73. Scholar
  4. 4.
    Harris KM, Perry E, Bourne J et al (2006) Uniform serial sectioning for transmission electron microscopy. J Neurosci 26:12101–12103. Scholar
  5. 5.
    Bock DD, Lee WC, Kerlin AM et al (2011) Network anatomy and in vivo physiology of visual cortical neurons. Nature 2011(471):177–182. Scholar
  6. 6.
    Hayworth KJ, Kasthuri N, Schalek R et al (2006) Automating the collection of ultrathin serial sections for large volume TEM reconstructions. Microsc Microanal 12:86–87. Scholar
  7. 7.
    Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2:e329. Scholar
  8. 8.
    Knott G, Marchman H, Wall D et al (2008) Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J Neurosci 28:2959–2964. Scholar
  9. 9.
    Scheffer LK, Meinertzhagen IA (2019) The fly brain atlas. Annu Rev Cell Dev Biol 35:737–653. Scholar
  10. 10.
    Takemura SY, Bharioke A, Lu Z et al (2013) A visual motion detection circuit suggested by Drosophila connectomics. Nature 500:175–181.
  11. 11.
    Xu CS, Januszewski M, Lu Z et al (2020) A connectome of the adult Drosophila central brain. bioRxiv:2020.01.21.911859.
  12. 12.
    Scheffer LK, Xu CS, Januszewski M et al (2020) A connectome and analysis of the adult Drosophila central brain. bioRxiv:2020.04.07.030213. Scholar
  13. 13.
    Januszewski M, Kornfeld J, Li PH et al (2018) High-precision automated reconstruction of neurons with flood-filling networks. Nat Methods 15:605–610. Scholar
  14. 14.
    Meinertzhagen IA (2016) Connectome studies on Drosophila: a short perspective on a tiny brain. J Neurogenet 30:62–68.
  15. 15.
    Schneider-Mizell CM, Gerhard S, Longair M et al (2016) Quantitative neuroanatomy for connectomics in Drosophila. eLife 5:e12059. Scholar
  16. 16.
    Helmstaedter M (2013) Cellular-resolution connectomics: challenges of dense neural circuit reconstruction. Nat Methods 10(6):501–507. Scholar
  17. 17.
    Takemura SY, Xu CS, Lu Z et al (2015) Synaptic circuits and their variations within different columns in the visual system of Drosophila. PNAS 112:13711–13716. Scholar
  18. 18.
    Shinomiya K, Huang G, Lu Z et al. (2019) Comparisons between the ON- and OFF-edge motion pathways in the Drosophila brain. eLife 8:e40025. doi:
  19. 19.
    Takemura S, Aso Y, Hige T et al (2017) A connectome of a learning and memory center in the adult Drosophila brain. eLife 6:e26975. Scholar
  20. 20.
    Horne JA, Langille C, McLin S et al. (2018) A resource for the Drosophila antennal lobe provided by the connectome of glomerulus VA1v. eLife 7:e37500. doi:
  21. 21.
    Titze B (2013) Techniques to prevent sample surface charging and reduce beam damage effects for SBEM imaging. Dissertation, Heidelberg University, pp 1–112Google Scholar
  22. 22.
    Calcagno L, Compagnini G, Foti G (1992) Structural modification of polymer films by ion irradiation. Nucl Instrum Methods Phys Res, Sect B 65(1–4):413–422. Scholar
  23. 23.
    Hayworth KJ, Xu CS, Lu Z et al (2015) Ultrastructurally smooth thick partitioning and volume stitching for large-scale connectomics. Nat Methods 12:319–322.
  24. 24.
    McGee-Russell SM, De Bruijn WC, Gosztonyi G (1990) Hot knife microtomy for large area sectioning and combined light and electron microscopy in neuroanatomy and neuropathology. J Neurocytol 19(5):655–661. Scholar
  25. 25.
    Lu Z, Xu CS, Hayworth KJ et al (2019) En bloc preparation of Drosophila brains enables high-throughput FIB-SEM connectomics. bioRxiv:855130.
  26. 26.
    Gao R, Asano SM, Upadhyayula S et al (2019) Cortical column and whole-brain imaging with molecular contrast and nanoscale resolution. Science 363(6424):eaau8302. Scholar
  27. 27.
    Ioannou S, Jackson J, Sheu S et al (2019) Neuron-astrocyte metabolic coupling protects against activity-induced fatty acid toxicity. Cell 177(6):1522–1535.
  28. 28.
    Nixon-Abell J, Obara CJ, Weigel AV et al (2016) Increased spatiotemporal resolution reveals highly dynamic dense tubular matrices in the peripheral. Science 354(6311):433–446. Scholar
  29. 29.
    Hoffman DP, Shtengel G, Xu CS et al (2019) Correlative three-dimensional super-resolution and block face electron microscopy of whole vitreously frozen cells. Science 367 (6475):eaaz5357. 10.1101/773986Google Scholar
  30. 30.
    Hennig P, Denk W (2007) Point-spread functions for backscattered imaging in the scanning electron microscope. J App Phys 102:123101–123108. Scholar
  31. 31.
    Wu Y, Whiteus C, Xu CS et al (2017) Contacts between the endoplasmic reticulum and other membranes in neurons. PNAS 114(24):E4859–E4867.
  32. 32.
    Hua Y, Laserstein P, Helmstaedter M (2015) Large-volume en-bloc staining for electron microscopy-based connectomics. Nat Commun 6:7923. Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • C. Shan Xu
    • 1
    Email author
  • Song Pang
    • 1
  • Kenneth J. Hayworth
    • 1
  • Harald F. Hess
    • 1
  1. 1.Howard Hughes Medical InstituteAshburnUSA

Personalised recommendations