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Focused ion beams in biology

Abstract

A quiet revolution is under way in technologies used for nanoscale cellular imaging. Focused ion beams, previously restricted to the materials sciences and semiconductor fields, are rapidly becoming powerful tools for ultrastructural imaging of biological samples. Cell and tissue architecture, as preserved in plastic-embedded resin or in plunge-frozen form, can be investigated in three dimensions by scanning electron microscopy imaging of freshly created surfaces that result from the progressive removal of material using a focused ion beam. The focused ion beam can also be used as a sculpting tool to create specific specimen shapes such as lamellae or needles that can be analyzed further by transmission electron microscopy or by methods that probe chemical composition. Here we provide an in-depth primer to the application of focused ion beams in biology, including a guide to the practical aspects of using the technology, as well as selected examples of its contribution to the generation of new insights into subcellular architecture and mechanisms underlying host-pathogen interactions.

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Figure 1: 3D imaging of large biological samples by FIB-SEM.
Figure 2: Visualizing cells and cell-cell contacts in three dimensions.
Figure 3: Imaging the large and small by FIB-SEM.
Figure 4: 3D imaging of specific targets with correlative LM and FIB-SEM.
Figure 5: FIB thinning and lift-out of specimens for cryo-imaging.
Figure 6: FIBs in chemical imaging.

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References

  1. Ackerman, M.J., Spitzer, V.M., Scherzinger, A.L. & Whitlock, D.G. The Visible Human data set: an image resource for anatomical visualization. Medinfo 8, 1195–1198 (1995).

    PubMed  Google Scholar 

  2. Subramaniam, S. Bridging the imaging gap: visualizing subcellular architecture with electron tomography. Curr. Opin. Microbiol. 8, 316–322 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Spacek, J. & Lieberman, A.R. Ultrastructure and three-dimensional organization of synaptic glomeruli in rat somatosensory thalamus. J. Anat. 117, 487–516 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Thaemert, J.C. Ultrastructural interrelationships of nerve processes and smooth muscle cells in three dimensions. J. Cell Biol. 28, 37–49 (1966).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. White, J.G., Southgate, E., Thomson, J.N. & Brenner, S. The structure of the nervous system of the nematode Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 314, 1–340 (1986).

    Article  CAS  PubMed  Google Scholar 

  6. Baumeister, W., Grimm, R. & Walz, J. Electron tomography of molecules and cells. Trends Cell Biol. 9, 81–85 (1999).

    Article  CAS  PubMed  Google Scholar 

  7. Bartesaghi, A. et al. Classification and 3D averaging with missing wedge correction in biological electron tomography. J. Struct. Biol. 162, 436–450 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cantele, F., Zampighi, L., Radermacher, M., Zampighi, G. & Lanzavecchia, S. Local refinement: an attempt to correct for shrinkage and distortion in electron tomography. J. Struct. Biol. 158, 59–70 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Gan, L. & Jensen, G.J. Electron tomography of cells. Q. Rev. Biophys. 45, 27–56 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Chang, J., Liu, X., Rochat, R.H., Baker, M.L. & Chiu, W. Reconstructing virus structures from nanometer to near-atomic resolutions with cryo-electron microscopy and tomography. Adv. Exp. Med. Biol. 726, 49–90 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lučič, V., Rigort, A. & Baumeister, W. Cryo-electron tomography: the challenge of doing structural biology in situ. J. Cell Biol. 202, 407–419 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Briggs, J.A. Structural biology in situ—the potential of subtomogram averaging. Curr. Opin. Struct. Biol. 23, 261–267 (2013).

    Article  CAS  PubMed  Google Scholar 

  13. Noske, A.B., Costin, A.J., Morgan, G.P. & Marsh, B.J. Expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets. J. Struct. Biol. 161, 298–313 (2008).

    Article  PubMed  Google Scholar 

  14. Soto, G.E. et al. Serial section electron tomography: a method for three-dimensional reconstruction of large structures. Neuroimage 1, 230–243 (1994).

    Article  CAS  PubMed  Google Scholar 

  15. Micheva, K.D. & Smith, S.J. Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55, 25–36 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hayworth, K.J. et al. Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits. Front. Neural Circuits 8, 68 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  17. Denk, W. & Horstmann, H. Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2, e329 (2004). This paper describes the idea of putting an adapted microtome inside a scanning electron microscope to obtain automated serial sections; also see refs. 19 and 20.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Tapia, J.C. et al. High-contrast en bloc staining of neuronal tissue for field emission scanning electron microscopy. Nat. Protoc. 7, 193–206 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Helmstaedter, M. et al. Connectomic reconstruction of the inner plexiform layer in the mouse retina. Nature 500, 168–174 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Briggman, K.L., Helmstaedter, M. & Denk, W. Wiring specificity in the direction-selectivity circuit of the retina. Nature 471, 183–188 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Briggman, K.L. & Denk, W. Towards neural circuit reconstruction with volume electron microscopy techniques. Curr. Opin. Neurobiol. 16, 562–570 (2006).

    Article  CAS  PubMed  Google Scholar 

  22. Seligman, A.M., Wasserkrug, H.L. & Hanker, J.S. A new staining method (OTO) for enhancing contrast of lipid-containing membranes and droplets in osmium tetroxide–fixed tissue with osmiophilic thiocarbohydrazide(TCH). J. Cell Biol. 30, 424–432 (1966).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Adams, J.C. Heavy metal intensification of DAB-based HRP reaction product. J. Histochem. Cytochem. 29, 775 (1981).

    Article  CAS  PubMed  Google Scholar 

  24. Kizilyaprak, C., Longo, G., Daraspe, J. & Humbel, B.M. Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy. J. Struct. Biol. 189, 135–146 (2015).

    Article  CAS  PubMed  Google Scholar 

  25. Livengood, R.H. et al. The neon gas field ion source—a first characterization of neon nanomachining properties. Nucl. Instrum. Methods Phys. Res. A 645, 136–140 (2011).

    Article  CAS  Google Scholar 

  26. Smith, N.S. et al. High brightness inductively coupled plasma source for high current focused ion beam applications. J. Vac. Sci. Technol. B Nanotechnol. Microelectron. 24, 2902–2906 (2006).

    Article  CAS  Google Scholar 

  27. Holzer, L., Indutnyi, F., Gasser, P.H., Munch, B. & Wegmann, M. Three-dimensional analysis of porous BaTiO3 ceramics using FIB nanotomography. J. Microsc. 216, 84–95 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Orloff, J. High-resolution focused ion-beams. Rev. Sci. Instrum. 64, 1105–1130 (1993).

    Article  CAS  Google Scholar 

  29. Burdet, P., Vannod, J., Hessler-Wyser, A., Rappaz, M. & Cantoni, M. Three-dimensional chemical analysis of laser-welded NiTi-stainless steel wires using a dual-beam FIB. Acta Mater. 61, 3090–3098 (2013).

    Article  CAS  Google Scholar 

  30. Inkson, B.J., Steer, T., Mobus, G. & Wagner, T. Subsurface nanoindentation deformation of Cu-Al multilayers mapped in 3D by focused ion beam microscopy. J. Microsc. 201, 256–269 (2001).

    Article  CAS  PubMed  Google Scholar 

  31. Keller, L.M. et al. Characterization of multi-scale microstructural features in Opalinus Clay. Microporous Mesoporous Mater. 170, 83–94 (2013).

    Article  CAS  Google Scholar 

  32. Volkert, C.A. & Minor, A.M. Focused ion beam microscopy and micromachining. MRS Bull. 32, 389–395 (2007).

    Article  CAS  Google Scholar 

  33. Fu, Y.Q., Kok, N. & Bryan, A. Microfabrication of microlens array by focused ion beam technology. Microelectron. Eng. 54, 211–221 (2000).

    Article  CAS  Google Scholar 

  34. Vasile, M.J., Nassar, R., Xie, J. & Guo, H. Microfabrication techniques using focused ion beams and emergent applications. Micron 30, 235–244 (1999).

    Article  CAS  PubMed  Google Scholar 

  35. Giannuzzi, L.A., Drown, J.L., Brown, S.R., Irwin, R.B. & Stevie, F. Applications of the FIB lift-out technique for TEM specimen preparation. Microsc. Res. Tech. 41, 285–290 (1998).

    Article  CAS  PubMed  Google Scholar 

  36. Giannuzzi, L.A. Routine backside FIB milling with EXpressLO (TM). In Proc. 38th International Symposium for Testing and Failure Analysis (ISTFA 2012) 388–390 (ASM International, 2012).

    Google Scholar 

  37. Bassim, N., Scott, K. & Giannuzzi, L.A. Recent advances in focused ion beam technology and applications. MRS Bull. 39, 317–325 (2014).

    Article  CAS  Google Scholar 

  38. Cantoni, M. & Holzer, L. Advances in 3D focused ion beam tomography. MRS Bull. 39, 354–360 (2014). References 37 and 38 are good reviews that describe basic aspects of the physics of FIBs and their use, primarily in materials sciences.

    Article  CAS  Google Scholar 

  39. Hayworth, K.J. et al. Ultrastructurally smooth thick partitioning and volume stitching for large-scale connectomics. Nat. Methods 12, 319–322 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kopek, B.G., Shtengel, G., Xu, C.S., Clayton, D.A. & Hess, H.F. Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes. Proc. Natl. Acad. Sci. USA 109, 6136–6141 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. De Winter, D.A.M. et al. Tomography of insulating biological and geological materials using focused ion beam (FIB) sectioning and low-kV BSE imaging. J. Microsc. 233, 372–383 (2009).

    Article  PubMed  Google Scholar 

  42. Murphy, G.E. et al. Correlative 3D imaging of whole mammalian cells with light and electron microscopy. J. Struct. Biol. 176, 268–278 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Hildebrand, M., Kim, S., Shi, D., Scott, K. & Subramaniam, S. 3D imaging of diatoms with ion-abrasion scanning electron microscopy. J. Struct. Biol. 166, 316–328 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Scott, K. & Ritchie, N.W.M. Analysis of 3D elemental mapping artefacts in biological specimens using Monte Carlo simulation. J. Microsc. 233, 331–339 (2009).

    Article  CAS  PubMed  Google Scholar 

  45. Wang, K., Strunk, K., Zhao, G.P., Gray, J.L. & Zhang, P.J. 3D structure determination of native mammalian cells using cryo-FIB and cryo-electron tomography. J. Struct. Biol. 180, 318–326 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Marko, M., Hsieh, C., Schalek, R., Frank, J. & Mannella, C. Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nat. Methods 4, 215–217 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. Rigort, A. et al. Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography. Proc. Natl. Acad. Sci. USA 109, 4449–4454 (2012). References 46 and 47 describe approaches for using the FIB to thin cryo-specimens for high-resolution TEM imaging.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Knott, G., Rosset, S. & Cantoni, M. Focussed ion beam milling and scanning electron microscopy of brain tissue. J. Vis. Exp. 2011, e2588 (2011).

    Google Scholar 

  49. Bushby, A.J. et al. Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy. Nat. Protoc. 6, 845–858 (2011).

    Article  CAS  PubMed  Google Scholar 

  50. Heymann, J.A. et al. 3D imaging of mammalian cells with ion-abrasion scanning electron microscopy. J. Struct. Biol. 166, 1–7 (2009).

    Article  CAS  PubMed  Google Scholar 

  51. Heymann, J.A. et al. Site-specific 3D imaging of cells and tissues with a dual beam microscope. J. Struct. Biol. 155, 63–73 (2006). This is the first report of the use of FIB-SEM imaging to obtain a 3D-volume reconstruction of a cell or tissue specimen.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Bennett, A.E. et al. Ion-abrasion scanning electron microscopy reveals surface-connected tubular conduits in HIV-infected macrophages. PLoS Pathog. 5, e1000591 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Van Voorhis, W.C., Hair, L.S., Steinman, R.M. & Kaplan, G. Human dendritic cells. Enrichment and characterization from peripheral blood. J. Exp. Med. 155, 1172–1187 (1982).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Felts, R.L. et al. 3D visualization of HIV transfer at the virological synapse between dendritic cells and T cells. Proc. Natl. Acad. Sci. USA 107, 13336–13341 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Do, T. et al. Three-dimensional imaging of HIV-1 virological synapses reveals membrane architectures involved in virus transmission. J. Virol. 88, 10327–10339 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Armer, H.E. et al. Imaging transient blood vessel fusion events in zebrafish by correlative volume electron microscopy. PLoS One 4, e7716 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Murphy, G.E. et al. Ion-abrasion scanning electron microscopy reveals distorted liver mitochondrial morphology in murine methylmalonic acidemia. J. Struct. Biol. 171, 125–132 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Schneider, P., Meier, M., Wepf, R. & Muller, R. Serial FIB/SEM imaging for quantitative 3D assessment of the osteocyte lacuno-canalicular network. Bone 49, 304–311 (2011).

    Article  PubMed  Google Scholar 

  59. Glancy, B. et al. Mitochondrial reticulum for cellular energy distribution in muscle. Nature 523, 617–620 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Narayan, K. et al. Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology. J. Struct. Biol. 185, 278–284 (2014). This paper describes some of the recent advances in increasing the scope and robustness of FIB-SEM imaging of biological samples.

    Article  CAS  PubMed  Google Scholar 

  61. Schertel, A. et al. Cryo FIB-SEM: volume imaging of cellular ultrastructure in native frozen specimens. J. Struct. Biol. 184, 355–360 (2013). This study describes cryo–FIB-SEM, in which biological samples are FIB-milled and imaged by the SEM at cryogenic temperatures.

    Article  CAS  PubMed  Google Scholar 

  62. Villinger, C. et al. FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells. Histochem. Cell Biol. 138, 549–556 (2012).

    Article  CAS  PubMed  Google Scholar 

  63. Stell, W.K. Correlated light + electron microscope observations on Golgi preparations of goldfish retina. J. Cell Biol. 23, 89A (1964).

    Article  Google Scholar 

  64. Hanker, J.S., Deb, C., Wasserkrug, H.L. & Seligman, A.M. Staining tissue for light and electron microscopy by bridging metals with multidentate ligands. Science 152, 1631–1634 (1966).

    Article  CAS  PubMed  Google Scholar 

  65. Seligman, A.M., Ueno, H., Wasserkrug, H.L. & Hanker, J.S. Esterase method for light and electron microscopy via the formation of osmiophilic diazothioethers (1, 2). Ann. Histochim. 11, 115–129 (1966).

    CAS  PubMed  Google Scholar 

  66. de Boer, P., Hoogenboom, J.P. & Giepmans, B.N. Correlated light and electron microscopy: ultrastructure lights up! Nat. Methods 12, 503–513 (2015).

    Article  CAS  PubMed  Google Scholar 

  67. Zhang, P. Correlative cryo-electron tomography and optical microscopy of cells. Curr. Opin. Struct. Biol. 23, 763–770 (2013).

    Article  CAS  PubMed  Google Scholar 

  68. Modla, S. & Czymmek, K.J. Correlative microscopy: a powerful tool for exploring neurological cells and tissues. Micron 42, 773–792 (2011).

    Article  CAS  PubMed  Google Scholar 

  69. Sjollema, K.A., Schnell, U., Kuipers, J., Kalicharan, R. & Giepmans, B.N. Correlated light microscopy and electron microscopy. Methods Cell Biol. 111, 157–173 (2012).

    Article  CAS  PubMed  Google Scholar 

  70. Hanein, D. & Volkmann, N. Correlative light-electron microscopy. Adv. Protein Chem. Struct. Biol. 82, 91–99 (2011).

    Article  CAS  PubMed  Google Scholar 

  71. Rigort, A., Villa, E., Bauerlein, F.J., Engel, B.D. & Plitzko, J.M. Integrative approaches for cellular cryo-electron tomography: correlative imaging and focused ion beam micromachining. Methods Cell Biol. 111, 259–281 (2012).

    Article  CAS  PubMed  Google Scholar 

  72. Biel, S.S., Kawaschinski, K., Wittern, K.P., Hintze, U. & Wepf, R. From tissue to cellular ultrastructure: closing the gap between micro- and nanostructural imaging. J. Microsc. 212, 91–99 (2003).

    Article  CAS  PubMed  Google Scholar 

  73. Kukulski, W. et al. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision. J. Cell Biol. 192, 111–119 (2011). This paper describes an approach for getting extremely accurate correlation between LM and EM images of cryogenic samples.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Watanabe, S. et al. Protein localization in electron micrographs using fluorescence nanoscopy. Nat. Methods 8, 80–84 (2011).

    Article  CAS  PubMed  Google Scholar 

  75. Perkovic, M. et al. Correlative light- and electron microscopy with chemical tags. J. Struct. Biol. 186, 205–213 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Sosinsky, G.E., Giepmans, B.N., Deerinck, T.J., Gaietta, G.M. & Ellisman, M.H. Markers for correlated light and electron microscopy. Methods Cell Biol. 79, 575–591 (2007). This report provides a detailed list of markers and approaches available for CLEM.

    Article  CAS  PubMed  Google Scholar 

  77. Brown, E. & Verkade, P. The use of markers for correlative light electron microscopy. Protoplasma 244, 91–97 (2010).

    Article  PubMed  Google Scholar 

  78. Giepmans, B.N., Deerinck, T.J., Smarr, B.L., Jones, Y.Z. & Ellisman, M.H. Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots. Nat. Methods 2, 743–749 (2005).

    Article  CAS  PubMed  Google Scholar 

  79. Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507 (2002).

    Article  CAS  PubMed  Google Scholar 

  80. Shu, X. et al. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9, e1001041 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Grabenbauer, M. et al. Correlative microscopy and electron tomography of GFP through photooxidation. Nat. Methods 2, 857–862 (2005).

    Article  CAS  PubMed  Google Scholar 

  82. McDonald, K.L. A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues. J. Microsc. 235, 273–281 (2009). This review provides a detailed description of high-pressure freezing protocols used to avoid ultrastructural artifacts commonly seen with conventionally fixed biological samples.

    Article  CAS  PubMed  Google Scholar 

  83. Taylor, K.A. & Glaeser, R.M. Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future. J. Struct. Biol. 163, 214–223 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Koster, A.J. et al. Perspectives of molecular and cellular electron tomography. J. Struct. Biol. 120, 276–308 (1997).

    Article  CAS  PubMed  Google Scholar 

  85. McIntosh, R., Nicastro, D. & Mastronarde, D. New views of cells in 3D: an introduction to electron tomography. Trends Cell Biol. 15, 43–51 (2005).

    Article  CAS  PubMed  Google Scholar 

  86. Al-Amoudi, A. et al. Cryo-electron microscopy of vitreous sections. EMBO J. 23, 3583–3588 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  87. Harapin, J. et al. Structural analysis of multicellular organisms with cryo-electron tomography. Nat. Methods 12, 634–636 (2015).

    Article  CAS  PubMed  Google Scholar 

  88. Kamino, T., Yaguchi, T., Ohnishi, T., Ishitani, T. & Osumi, M. Application of a FIB-STEM system for 3D observation of a resin-embedded yeast cell. J. Electron Microsc. (Tokyo) 53, 563–566 (2004).

    Article  Google Scholar 

  89. Hsieh, C., Schmelzer, T., Kishchenko, G., Wagenknecht, T. & Marko, M. Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography. J. Struct. Biol. 185, 32–41 (2014).

    Article  CAS  PubMed  Google Scholar 

  90. Hayles, M.F. et al. The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy. J. Struct. Biol. 172, 180–190 (2010).

    Article  PubMed  Google Scholar 

  91. Evans, C.L. & Xie, X.S. Coherent anti-stokes Raman scattering microscopy: chemical imaging for biology and medicine. Annu. Rev. Anal. Chem. (Palo Alto Calif.) 1, 883–909 (2008).

    Article  CAS  Google Scholar 

  92. Petibois, C. Imaging methods for elemental, chemical, molecular, and morphological analyses of single cells. Anal. Bioanal. Chem. 397, 2051–2065 (2010).

    Article  CAS  PubMed  Google Scholar 

  93. Fletcher, J.S. & Vickerman, J.C. Secondary ion mass spectrometry: characterizing complex samples in two and three dimensions. Anal. Chem. 85, 610–639 (2013).

    Article  CAS  PubMed  Google Scholar 

  94. Ostrowski, S.G., Van Bell, C.T., Winograd, N. & Ewing, A.G. Mass spectrometric imaging of highly curved membranes during Tetrahymena mating. Science 305, 71–73 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Fletcher, J.S., Lockyer, N.P., Vaidyanathan, S. & Vickerman, J.C. TOF-SIMS 3D biomolecular imaging of Xenopus laevis oocytes using buckminsterfullerene (C-60) primary ions. Anal. Chem. 79, 2199–2206 (2007).

    Article  CAS  PubMed  Google Scholar 

  96. Szakal, C., Narayan, K., Fu, J., Lefman, J. & Subramaniam, S. Compositional mapping of the surface and interior of mammalian cells at submicrometer resolution. Anal. Chem. 83, 1207–1213 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. James, S.A. et al. Quantification of ZnO nanoparticle uptake, distribution, and dissolution within individual human macrophages. ACS Nano 7, 10621–10635 (2013).

    Article  CAS  PubMed  Google Scholar 

  98. Kelly, T.F. & Miller, M.K. Invited review article: atom probe tomography. Rev. Sci. Instrum. 78, 031101 (2007).

    Article  PubMed  CAS  Google Scholar 

  99. Allen, J.E. et al. High-resolution detection of Au catalyst atoms in Si nanowires. Nat. Nanotechnol. 3, 168–173 (2008).

    Article  CAS  PubMed  Google Scholar 

  100. Thompson, K. et al. In situ site-specific specimen preparation for atom probe tomography. Ultramicroscopy 107, 131–139 (2007).

    Article  CAS  PubMed  Google Scholar 

  101. Langford, R.M. & Clinton, C. In situ lift-out using a FIB-SEM system. Micron 35, 607–611 (2004).

    Article  CAS  PubMed  Google Scholar 

  102. Narayan, K., Prosa, T.J., Fu, J., Kelly, T.F. & Subramaniam, S. Chemical mapping of mammalian cells by atom probe tomography. J. Struct. Biol. 178, 98–107 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Stegmann, H., Dömer, H., Rosenkranz, R. & Zschech, E. Efficient target preparation by combined pulsed laser ablation and FIB milling. Microsc. Microanal. 17, 658–659 (2011).

    Article  Google Scholar 

  104. Smith, C. Microscopy: two microscopes are better than one. Nature 492, 293–297 (2012).

    Article  CAS  PubMed  Google Scholar 

  105. Liu, T., Jones, C., Seyedhosseini, M. & Tasdizen, T. A modular hierarchical approach to 3D electron microscopy image segmentation. J. Neurosci. Methods 226, 88–102 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  106. Nunez-Iglesias, J., Kennedy, R., Plaza, S.M., Chakraborty, A. & Katz, W.T. Graph-based active learning of agglomeration (GALA): a Python library to segment 2D and 3D neuroimages. Front. Neuroinform. 8, 34 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  107. Chklovskii, D.B., Vitaladevuni, S. & Scheffer, L.K. Semi-automated reconstruction of neural circuits using electron microscopy. Curr. Opin. Neurobiol. 20, 667–675 (2010). This report describes an approach for segmenting and reconstructing specific shapes (here, neurons) in a large 3D EM image volume.

    Article  CAS  PubMed  Google Scholar 

  108. Kremer, J.R., Mastronarde, D.N. & McIntosh, J.R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  PubMed  Google Scholar 

  109. Hartnell, L.M., Earl, L.A., Bliss, D., Moran, A. & Subramaniam, S. Imaging cellular architecture with 3D SEM. In Encyclopedia of Cell Biology Vol. 2 (Eds. Bradshaw, R. & Stahl, P.) 44–50 (Academic Press, 2016).

    Book  Google Scholar 

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Acknowledgements

This work was supported by funds from the intramural program of the National Cancer Institute, US National Institutes of Health, Bethesda, Maryland, USA. The authors thank E. Tyler for artistic rendering of the figures; E. He, A. Brust and D. Bliss for help creating Supplementary Video 1 describing the FIB-SEM imaging process; and L. Earl and other members of their laboratory for many fruitful discussions. The authors apologize to those colleagues whose work has not been cited owing to space constraints.

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Correspondence to Sriram Subramaniam.

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FIB-SEM imaging of biological samples (MOV 26161 kb)

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Narayan, K., Subramaniam, S. Focused ion beams in biology. Nat Methods 12, 1021–1031 (2015). https://doi.org/10.1038/nmeth.3623

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