Focused Ion Beam Technology as a Fabrication and Inspection Tool in Neuron Interfacing



Recent trends in the development of devices for electrophysiology involve the fabrication of electrodes with three-dimensional micro- and nanoprotrusions. These devices take advantage of the natural capacity of cells to actively interact with nanostructured substrates in order to realize a more intimate cell-to-electrode coupling. In this chapter, we review the use of focused ion beam (FIB) technology as a versatile tool for fabricating nanostructures of different shape and size on top of freely chosen substrates. This approach allows custom design and fabrication of nanoprotrusions to optimize cell-to-electrode electrical coupling, while at the same time allowing leeway to optimize the microelectronic substrate. Examples of enhanced interaction of cells with nanostructures are reviewed, with respect to nanoprotrusion geometry and surface functionalization, to illustrate the potential of FIB-based deposition as a tool for realizing new types of nanostructures for neurophysiological measurements. Finally, the combined focused ion beam/scanning electron microscope is presented as a tool for investigating the physical basis for interactions between neuronal cell membranes and nanostructured surfaces.


Electrical Coupling Extracellular Recording Multi Electrode Array Aplysia Neuron Nanostructured Substrate 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Hodgkin, A., Huxley, A.: A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. 117, 500–544 (1952).Google Scholar
  2. 2.
    Purves, R.D. Microelectrode Methods for Intracellular Recording and Ionophoresis. Academic press, London (1981)Google Scholar
  3. 3.
    Shahaf, G., and Marom, S.: Learning in networks of cortical neurons, J. Neurosci. 21, 8782–8788 (2001).Google Scholar
  4. 4.
    Shahaf, G., Eytan, D., Gal, A., Kermany, E., Lyakhov, V., Zrenner, C., Marom, S.: Order based representation in random networks of cortical neurons. PLoS Comput. Biol. 4, e1000228 (2008)MathSciNetGoogle Scholar
  5. 5.
    Bonifazi, P., Ruaro, M.E., Torre, V.: Statistical properties of information processing in neuronal networks. Eur. J. Neurosci. 22, 2953–2964 (2005)Google Scholar
  6. 6.
    Chiappalone, M., Bove, M., Vato, A., Tedesco, M., and Martinioia, S.: Dissociated cortical networks show spontaneously correlated activity patterns during in vitro development. Brain Res. 1093, 41–53 (2006)Google Scholar
  7. 7.
    Chiappalone, M., Vato, A., Tedesco, M., Marcoli, M., Davide, F., and Martinoia, S.: Networks of neurons coupled to microelectrode array: a neuronal sensory system for pharmacological applications. Biosens. Bioelectron. 18, 627–634 (2003)Google Scholar
  8. 8.
    Scelfo, B., Politi, M., Reniero, F., Palosaari, T., Whelan, M., Zaldívar, J-M.: Application of multielectrode array (MEA) chips for the evaluation of mixtures neurotoxicity. Toxicology 299, 172–183 (2012)Google Scholar
  9. 9.
    Gopal, K.V., Gross, G.W. Emerging histiotypic properties of cultured neuronal networks. In: Baudry, M., Taketani, M. (eds.) Advances in Network Electrophysiology Using Multi-Electrode Arrays, pp. 193–214. Springer, New York (2006)Google Scholar
  10. 10.
    Xia, Y., and Gross, G.W.: Histotypic electrophysiological responses of cultured neuronal networks to ethanol. Alcohol 30, 167–174 (2003)Google Scholar
  11. 11.
    Wise, K.D.: Silicon microsystems for neuroscience and neural prostheses. IEEE Eng. Med. Bio. Magazine 24, 22–29 (2005)Google Scholar
  12. 12.
    Normann, R.A.: Technology insight: future neuroprosthetic therapies for disorder of the nervous system. Nat. Clin. Pract. Neuro. 3, 444–452 (2007)Google Scholar
  13. 13.
    Buzsáki, G.: Large-scale recording of neuronal ensembles. Nat. Neurosc. 7, 446–451 (2004)Google Scholar
  14. 14.
    Huettel, S.A., Song, A.W., McCarthy, G.: Functional Magnetic Resonance Imaging, 2nd edn. Sinauer Associates, Massachusetts (2008)Google Scholar
  15. 15.
    Gold, C., Henze, D.A., Koch, C., and Buzsáki, G.: On the origin of the extracellular action potential waveform: a modeling study. J. Neurophysiol. 95, 3113–3128 (2006)Google Scholar
  16. 16.
    Buzsáki, G., Anastassiou, C. A., and Koch, C.: The origin of extracellular fields and currents – EEG, ECoG, LFP and spikes. Nat. Rev. Neurosc. 13, 407–420 (2012)Google Scholar
  17. 17.
    Pine, J.: Recording action potentials from cultured neurons with extracellular microcircuit electrodes. J. Neurosci. Methods 2, 19–31 (1980)Google Scholar
  18. 18.
    Oka, H., Shimono, K., Ogawa, R., Sugihara, H., and Taketani, M.: A new planar multielectrode array for extracellular recording: application to hippocampal acute slice. J. Neurosci. Methods 93, 61–67 (1999)Google Scholar
  19. 19.
    Grumet, A.E., Wyatt, J.L., and Rizzo, J.F.: Multi-electrode stimulation and recording in the isolated retina. J. Neurosci. Methods 101, 31–42 (2000)Google Scholar
  20. 20.
    Kim, J.H., Kang, G., Nam, Y., and Choi, Y.K.: Surface-modified microelectrode array with flake nanostructure for neural recording and stimulation. Nanotechnology 21, 85303 (2010)Google Scholar
  21. 21.
    Bruggemann, D., Wolfrum, B., Maybeck, V., Mourzina, Y., Jansen, M., Offenähusser, A.: Nanostructured gold microelectrodes for extracellular recording from electrogenic cells. Nanotechnology 22, 265104 (2011)Google Scholar
  22. 22.
    Shein, M., Greenbaum, A., Gabay, T., Sorkin, R., David-Pur, M., Ben-Jacob, E., Hanein, Y.: Engineered neuronal circuits shaped and interfaced with carbon nanotube microelectrode arrays. Biomed. Microdevices 11, 495–501 (2009)Google Scholar
  23. 23.
    Keefer, E.W., Botterman, B.R., Romero, M.I., Rossi, A.F., and Gross, G.W.: Carbon nanotube coating improves neuronal recordings. Nat. Nanotech. 3, 434–439 (2008)Google Scholar
  24. 24.
    Kim, D.H., Richardson-Burns, S.M., Hendricks, J.L., Sequera, C., Martin, D.C.: Effect of immobilized nerve growth factor on conductive polymers: electrical properties and cellular response. Adv. Funct. Mater. 17, 79–86 (2007)Google Scholar
  25. 25.
    Kim, Y.T., Haftel, V.K., Kumar, S., Bellamkonda, R.V.: The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve gaps. Biomaterials 29, 3117–3127 (2008)Google Scholar
  26. 26.
    Abidian, M.R., Kim, D.H., Martin, D.C.: Conducting-polymer nanotubes for controlled drug release, Adv. Mater. 18, 405–409 (2006)Google Scholar
  27. 27.
    Cui, X., Wiler, J., Dzaman, M., Altschuler, R. A., Martin, D. C.: In vivo studies of polypyrrole/peptide coated neural probes. Biomaterials 24, 777–787 (2003)Google Scholar
  28. 28.
    Cogan, S.F.: Neural stimulation and recording electrodes. Annu. Rev. Biomed. Eng. 10, 275–309 (2008)Google Scholar
  29. 29.
    Cai, N., Gong, Y., Chian, K.S., Chan, V., and Liao, K.: Adhesion dynamics of porcine esophageal fibroblasts on extracellular matrix protein-functionalized poly(lactic acid). Biomed. Mater. 3, 15014 (2008)Google Scholar
  30. 30.
    Wrobel, G., Höller, M., Ingebrandt, S., Dieluweit, S., Sommerhage, F., Bochem, H.P., and Offenhäusser, A.: Transmission electron microscopy study of the cell–sensor interface. J. R. Soc. Interface 5, 213–222 (2008)Google Scholar
  31. 31.
    Sniadecki, N.J., Desai, R.A., Ruiz, S.A., and Chen, C.S.: Nanotechnology for cell–substrate interactions. Ann. Biomed. Eng. 34, 59–74 (2006)Google Scholar
  32. 32.
    Spatz, J.P., and Geiger, B.: Molecular engineering of cellular environments: cell adhesion to nano-digital surfaces. Methods Cell Biol. 83, 89–111 (2007)Google Scholar
  33. 33.
    Nikkhah, M., Edalat, F., Manoucheri, S., Khademhosseini, A.: Engineering microscale topographies to control the cell-substrate interface. Biomaterials 33, 5230–5246 (2012)Google Scholar
  34. 34.
    Van Meerbergen, B., Raemaekers, T., Winters, K., Braeken, D., Bartic, C., Engelborghs, Y., Annaert, W., and Borghs, G.: On chip induced phagocytosis for improved neuronal cell adhesion. NSTI-Nanotech. 2, 107–110 (2006)Google Scholar
  35. 35.
    Van Meerbergen, B., Jans, K., Loo, J., Reekmans, G.G., Braeken, D., Seon-Ah, C., Bonroy, K., Maes, G., Borghs, G., Engelborghs, Y., Annaert, W., and Bartic, C.: Peptide-functionalized microfabricated structures for improved on-chip neuronal adhesion. Paper presented at the 30th annual international IEEE EMBS conference, Vancouver, British Columbia, Canada, 20–24 August 2008Google Scholar
  36. 36.
    Spira, M.E., Kamber, D., Dormann, A., Cohen, A., Bartic, C., Borghs, G., Langedijk, J.P.M., Yitzchaik, S., Shabthai, K., and Shappir, J.: Improved neuronal adhesion to the surface of electronic device by engulfment of protruding micro-nails fabricated on the chip surface. Paper presented at the 14th international conference on solid-state sensors, actuators and microsystems, Lyon, France, 10–14 June 2007Google Scholar
  37. 37.
    Hai, A., Dormann, A., Shappir, J., Yitzchaik, S., Bartic, C., Borghs, G., Langedijk, J.P.M., and Spira, M.E.: Spine-shaped gold protrusions improve the adherence and electrical coupling of neurons with the surface of micro-electronic devices. J. R. Soc. Interface 6, 1153–1165 (2009)Google Scholar
  38. 38.
    Hai, A., Kamber, D., Malkinson, G., Erez, H., Mazurski, N., Shappir, J. and Spira, M.E.: Changing gears from chemical adhesion of cells to flat substrata toward engulfment of micro-protrusions by active mechanisms. J. Neural Eng. 6, 066009 (2009)Google Scholar
  39. 39.
    Hai, A., Shappir, J., and Spira, M.E.: Long-term, multisite, parallel, in-cell recording and stimulation by an array of extracellular microelectrodes. J. Neurophysiol. 104, 559–568 (2010)Google Scholar
  40. 40.
    Fendyur, A., Mazurski, N., Shappir, J., and Spira, M.E.: Formation of essential ultrastructural interface between cultured hippocampal cells and gold mushroom-shaped MEA - toward “IN-CELL” recordings from vertebrate neurons. Front. Neuroeng. (2011). doi: 10.3389/fneng.2011.00014Google Scholar
  41. 41.
    Hai, A. and Spira, M.E.: On-chip electroporation, membrane repair dynamics and transient in-cell recordings by arrays of gold mushroom-shaped microelectrodes. Lab. Chip 12, 2865–2873 (2012)Google Scholar
  42. 42.
    Fendyur, A., and Spira, M.E.: Toward on-chip, in cell recordings from cultured cardiomyocytes by arrays of gold mushroom-shaped microelectrodes. Front. Neuroeng. (2012) doi: 10.3389/fneng.2012.00021Google Scholar
  43. 43.
    Robinson, J.T., Jorgolli, M., and Park, H.: Nanowire electrodes for high stimulation and measurements of neural circuits. Front. Neuroeng. (2013) doi: 10.3389/fncir.2013.00038Google Scholar
  44. 44.
    Robinson, J.T., Jorgolli, M., Shalek, A.K., Yoon, M-H., Gertner, R.S., Park, H.: Vertical nanowire electrode arrays as a scalable platform for intracellular interfacing to neuronal circuits. Nat Nanotechnol 7,180–184 (2012)Google Scholar
  45. 45.
    Xie, C., Lin, Z., Hanson, L., Cui, Y., Cui, B.: Intracellular recording of action potentials by nanopillar electroporation. Nat. Nanotechnol. 7, 185–190 (2012)Google Scholar
  46. 46.
    Duan, X., Gao, R., Xie, P., Cohen-Karni, T., Quing, Q., Choe, H.S., Tian, B., Jiang, X. and Lieber, C.M.: Intracellular recordings of action potentials by an extracellular nanoscale field-effect transistor. Nat. Nanotechnol. 7, 174–179 (2012)Google Scholar
  47. 47.
    Giannuzzi, L.A., Stevie, F.A.: Introduction to Focused Ion Beams: Instrumentation, Theory, Techniques, and Practice. Springer, New York (2005)Google Scholar
  48. 48.
    Volkert, C.A., and Minor, A.M.: Focused ion beam microscopy and micromachining. MRS Bulletin 32, 389–399 (2007)Google Scholar
  49. 49.
    Langford, R.M., Nellen, P.M., Gierak, J., and Fu, Y.: Focused ion beam micro- and nanoengineering. MRS Bulletin 32, 417–423 (2007)Google Scholar
  50. 50.
    Ali, M.Y., Hung, W., Yongqi, F.: A review of focused ion beam sputtering. Int. J. Precis. Eng. Man. 11, 157–170 (2010)Google Scholar
  51. 51.
    Xie, C., Hanson, L., Xie, W., Lin, Z., Cui, B., Cui, Y.: Noninvasive neuron pinning with nanopillar arrays. Nano Lett. 10, 4020–4024 (2010)Google Scholar
  52. 52.
    Martiradonna, L., Quarta, L., Sileo, L., Schertel, A., Maccione, A., Simi, A., Dante, S., Scarpellini, A., Berdondini, L., De Vittorio, M.: Beam induced deposition of 3D electrodes to improve coupling to cells Microelectron. Eng. 97, 365–368 (2013)Google Scholar
  53. 53.
    Sileo, L., Pisanello, F., Quarta, L., Maccione, A., Simi, A., Berdondini, L., De Vittorio, M., Martiradonna, L.: Electrical coupling of mammalian neurons to microelectrodes with 3D nanoprotrusions. Microelectron. Eng. (2013). doi:10.1016/j.mee.2013.03.152.Google Scholar
  54. 54.
    Vogel, V. and Sheetz, M.: Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell. Biol. 7, 265–275 (2006)Google Scholar
  55. 55.
    Sniadecki, N.J., Desai, R.A., Ruiz, S.A., Chen, C.S.: Nanotechnology for cell-substrate interactions. Ann. Biomed. Eng. 34, 59–74 (2006)Google Scholar
  56. 56.
    Previtali, S.C., Nodari, A., Taveggia, C., Pardini, C., Dina, G., Villa, A., Wrabetz, L., Quattrini, A., Feltri, M.L.: Expression of laminin receptors in Schwann cell differentiation: evidence for distinct roles. J. Neurosci. 23, 5520–5530 (2003)Google Scholar
  57. 57.
    Clegg, D.O., Wingerd, K.L., Hikita, S.T., Tolhurst, E.C.: Integrins in the development, function and dysfunction of the nervous system. Front. Biosci. 8, 723–750 (2003)Google Scholar
  58. 58.
    Qian, L., Saltzman, W.M.: Improving the expansion and neuronal differentiation of mesenchymal stem cells through culture surface modification. Biomaterials 25, 1330–1337 (2004)Google Scholar
  59. 59.
    Mao, Y., Schwarzbauer, J.E.: Fibronectin fibrillogenesis, a cell-mediated matrix assembly process. Matrix Biol. 24, 389–399 (2005)Google Scholar
  60. 60.
    Rohr, S., Flückiger-Labrada, R., Kucera, J.P.: Photolithographically defined deposition of attachment factors as a versatile method for patterning the growth of different cell types in culture. Eur. J. Physiol. 446, 125–132 (2003)Google Scholar
  61. 61.
    Glass, J., Blevitt, J., Dickerson, K., Pierschbacher, M.D., Craig, W.S.: Cell attachment and motility on materials modified by surface-active RGD-containing peptides. Ann. N. Y. Acad. Sci. 745, 177–186 (1994)Google Scholar
  62. 62.
    Heiduschka, P., Gopel, W., Beck, W., Kraas, W., Kienle, S., Jung, G.: Microstructured peptide-functionalized surfaces by electrochemical polymerization. Chem. Eur. J. 2, 667–672 (1996)Google Scholar
  63. 63.
    Sorribas, H., Braun, D., Leder, L., Sonderegger, P., Tiefenauer, L.: Adhesion proteins for a tight neuron-electrode contact. J. Neurosci. Meth. 104, 122–141 (2001)Google Scholar
  64. 64.
    Romanova, E.V., Oxley, S.P., Rubakhin, S.S., Bohn, P.W., Sweedler, J.V.: Self-assembled monolayers of alkanethiols on gold modulate electrophysiological parameters and cellular morphology of cultured neurons. Biomaterials 27, 1665–1669 (2006)Google Scholar
  65. 65.
    Richert, L., Schneider, A., Vautier, D., Vodouhe, C., Jessel, N., Payan, E., Schaaf, P., Voegel, J.C., Picart, C.: Imaging cell interactions with native and cross-linked polyelectrolyte multilayers. Cell Biochem. Biophys. 44, 273–285 (2006)Google Scholar
  66. 66.
    Lambacher, A., and Fromherz, P.: Fluorescence interference contrast microscopy on oxidized silicon using a monomolecular dye layer. Appl. Phys. A 63, 207–216 (1996)Google Scholar
  67. 67.
    Braun, D., and Fromherz, P.: Fluorescence interference contrast microscopy of cell adhesion on oxidized silicon. Appl. Phys. A 65, 341–348 (1997)Google Scholar
  68. 68.
    Iwanaga, Y., Braun, D., and Fromherz, P.: No correlation of focal contacts and close adhesion by comparing GFP vinculin and fluorescence interference of Dil. Eur. Biophys. J. 30, 17–26 (2001)Google Scholar
  69. 69.
    Gleixner, R., Fromherz, P.: The extracellular electrical resistivity in cell adhesion. Biophys. J. 90, 2600–2611 (2006)Google Scholar
  70. 70.
    Brittinger, M., and Fromherz, P.: Field-effect transistor with recombinant potassium channels: fast and slow response by electrical and chemical interactions. Appl. Phys. A 81, 439–447 (2005)Google Scholar
  71. 71.
    Zeck, G., Fromherz, P.: Repulsion and attraction by extracellular matrix protein in cell adhesion studied with nerve cells and lipid vesicles on silicon chips. Langmuir 19, 1580–1585 (2003)Google Scholar
  72. 72.
    Greve, F., Frerker, S., Bittermann, A.G., Burkhardt, C., Hierlemann, A., Hall, H.: Molecular design and characterization of the neuron-microelectrode array interface. Biomaterials 28, 5246–5258 (2007)Google Scholar
  73. 73.
    Martinelli, V., Cellot, G., Toma, F.M., Long, C.S., Caldwell, J.H., Zentilin, L., Giacca, M., Turco, A., Prato, M., Ballerini, L., Mestroni, L.: Carbon nanotubes promote growth and spontaneous electrical activity in cultured cardiac myocytes. Nano Lett. 12, 1831–1838 (2012)Google Scholar
  74. 74.
    Bareket-Keren, L., and Hanein, Y.: Carbon nanotube-based multielectrode arrays for neuronal interfacing: progress and prospects. Front. Neuroeng. (2013) doi: 10.3389/fncir.2012.00122Google Scholar
  75. 75.
    Kotov, N.A., Winter, J.O., Clements, I.P., Jan, E., Timko, B.P., Campidelli, S., Pathak, S., Mazzatenta, A., Lieber, C.M., Prato, M., Bellamkonda, R.V., Silva, G.A., Wong Shi Kam, N., Patolsky, F., and Ballerini, L. (2009) Nanomaterials for neural interfaces. Adv. Mater. 21, 3970–4004Google Scholar
  76. 76.
    Heim, M., Yvert, B., Kuhn, A.: Nanostructuration strategies to enhance microelectrode array (MEA) performance for neuronal recording and stimulation. J. Physiol.-Paris 106, 137–145 (2012)Google Scholar
  77. 77.
    M. Jenkner, and P. Fromherz, Bistability of membrane conductance in cell adhesion observed in a neuron transistor. Phys. Rev. Lett. 79, 4705–4708 (1997)Google Scholar
  78. 78.
    Hanson, L., Lin, Z.C., Xie, C., Cui, Y. and Cui, B.: Characterization of the cell–nanopillar interface by transmission electron microscopy. Nano Lett. 12, 5815–5820 (2012)Google Scholar
  79. 79.
    Shalek, A.K., Robinson, J.T., Karp, E.S., Lee, J.S., Ahn, D.R., Yoon, M.H., Sutton, A., Jorgolli, M., Gertner, R.S., Gujral, T.S., MacBeath, G., Yang, E.G., Park, H.: Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells. Proc. Natl. Acad. Sci. U. S. A., 107, 1870–1875 (2010)Google Scholar
  80. 80.
    Vandersarl, J.J., Xu, A.M., Melosh, N.A.: Nanostraws for direct fluidic intracellular access. Nano Lett. 12, 3881–3886 (2012)Google Scholar
  81. 81.
    Mann, D.G.J., McKnight, T.E., McPherson, J.T., Hoyt, P.R., Melechko, A.V., Simpson, M.L., Sayler, G.S.: Inducible RNA interference-mediated gene silencing using nanostructured gene delivery arrays. ACS Nano 2, 69–76 (2008)Google Scholar
  82. 82.
    Tian, B., Cohen-Karni, T., Qing, Q., Duan, X., Xie, P., Lieber, C.M.: Three-dimensional, flexible nanoscale field-effect transistors as localized bioprobes. Science 329, 831–834 (2010)Google Scholar
  83. 83.
    Gingell, D., Todd, I., and Bailey, J.: Topography of cell glass apposition revealed by total internal-reflection fluorescence of volume markers. J. Cell Biol. 100, 1334–1338 (1985)Google Scholar
  84. 84.
    Berthing, T., Bonde, S., Sørensen, C.B., Utko, P., Nygård, J., Martinez, K.L.: Intact mammalian cell function on semiconductor nanowire arrays: new perspectives for cell-based biosensing. Small 7, 640–647 (2011)Google Scholar
  85. 85.
    Yang, P., Yan, R., Fardy, M.: Semiconductor nanowire: what’s next? Nano Lett. 10, 1529–1536 (2010)Google Scholar
  86. 86.
    Hällström, W., Mårtensson, T., Prinz, C., Gustavsson, P., Montelius, L., Samuelson, L., and Kanje, M.: Gallium phosphide nanowires as a substrate for cultured neurons. Nano Lett. 7, 2960–2965 (2007)Google Scholar
  87. 87.
    Bittermann, A. G., Burkhardt, C. and Hall, H.: Imaging of cell-to-material interfaces by SEM after in situ focused ion beam milling on flat surfaces and complex 3D-fibrous structures. Adv. Eng. Mater. 11, B182–B188 (2009)Google Scholar
  88. 88.
    Murphy, G.E., Narayan, K., Lowekamp, B.C., Hartnell, L.M., Heymann, J.A.W., Fu, J., Subramniam, S.: Correlative 3D imaging of whole mammalian cells with light and electron microscopy. J. Struct. Biol. 176, 268–278 (2011)Google Scholar
  89. 89.
    Edwards, R.L., Coles, G., Sharpe, W.N.: Comparison of tensile and bulge tests for thin-film silicon nitride. Exp. Mech. 44, 49–54 (2004)Google Scholar
  90. 90.
    Lamers E., Walboomers, X.F., Domanski, M., McKerr, G., O’Hagan, B.M., Barnes, C.A., Peto, L., Luttge, R., Winnubst, L.A.J.A., Gardeniers, H.J.G.E., and Jansen, J.A.: Cryo DualBeam focused ion beam–scanning electron microscopy to evaluate the interface between cells and nanopatterned scaffolds. Tissue Eng. Part C Methods 17, 1–7 (2011)Google Scholar
  91. 91.
    Volkert, C.A., Busch, S., Heiland, B., and Dehm, G.: Transmission electron microscopy of fluorapatite-gelatine composite particles prepared using focused ion beam milling. J. Microscopy 214, 208–212 (2004)MathSciNetGoogle Scholar
  92. 92.
    Dalby, M.J., Riehle, M.O., Sutherland, D.S., Agheli, H., Curtis, A.S.G.: Changes in fibroblast morphology in response to nano-columns produced by colloidal lithography. Biomaterials 25, 5415–5422 (2004)Google Scholar
  93. 93.
    Friedmann, A., Hoess, A., Cismak, A., Heilmann, A.: Investigation of cell-substrate interactions by focused ion beam preparation and scanning electron microscopy. Acta Biomater. 7, 2499–2507 (2011).Google Scholar
  94. 94.
    Wierzbicki, R., Købler, C., Jensen, M.R.B., Łopacińska, J., Schmidt, M., Skolimowski, M., Abeille, F., Qvortrup, K., Mølhave, K.: Mapping the complex morphology of cell interactions with nanowire substrates using FIB-SEM. PLoS ONE 8, e53307 (2013)Google Scholar
  95. 95.
    Martinez, E., Engel, E., Lopez-Iglesias, C., Mills, C.A., Planell, J.A., Samitier, J.: Focused ion beam/scanning electron microscopy characterization of cell behavior on polymer micro-nanopatterned substrates: a study of cell–substrate interactions. Micron 39, 11–116 (2008)Google Scholar
  96. 96.
    Lešer, V., Drobne, D., Pipan, Ž., Milani, M., Tatti, F.: Comparison of different preparation methods of biological samples for milling and SEM investigation. J. Microsc. 233, 309–319 (2009)MathSciNetGoogle Scholar
  97. 97.
    Edwards, H.K., Fay, M.W., Anderson, S.I., Scotchford, C.A., Grant, D.M., and Brown, P.D.: An appraisal of ultramicrotomy, FIBSEM and cryogenic FIBSEM techniques for the sectioning of biological cells on titanium substrates for TEM investigation. J. Microsc. 234, 16–25 (2009)MathSciNetGoogle Scholar
  98. 98.
    Heymann, J.A.W., Hayles, M., Gestmann, I., Giannuzzi, L.A., Lich, B., and Subramniam, S.: Site-specific 3D imaging of cells and tissues with a dual beam microscope. J. Struct. Biol. 155, 63–73 (2006)Google Scholar
  99. 99.
    Nordestgaard, B.G., Rostgaard, J.: Critical-point drying versus freeze drying for scanning electron microscopy: a quantitative and qualitative study on isolated hepatocytes. J. Microsc. 137, 189–207 (1985)Google Scholar
  100. 100.
    Marko, M., Hsieh, C., Shalek, R., Frank, J., and Mannella, C.: Focused-ion-beam thinning of frozen hydrated biological specimens for cryo-electron microscopy. Nat. Methods 4, 215–217 (2007)Google Scholar
  101. 101.
    Hayles, M.F., Stokes, D.J., Phifer, D., and Findlay, K.C.: A technique for improved focused ion beam milling of cryoprepared life science specimens. J. Microsc. 226, 263–269 (2007)MathSciNetGoogle Scholar
  102. 102.
    JiméNez, N., Van Donselaar, E.G., De Winter, D.A.M., Vocking, K., Verkleij, A.J., Post, J.A., Gridded Aclar: preparation methods and use for correlative light and electron microscopy of cell monolayers, by TEM and FIB-SEM. J. Microsc. 237, 208–220 (2010)MathSciNetGoogle Scholar
  103. 103.
    Knott, G., Marchman, H., Wall, D., Lich, B.: Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J. Neurosc. 28, 2959–2964 (2008)Google Scholar
  104. 104.
    Gnauck, P., Burkhardt, C., Wolburg, H., Nisch, W.: Investigation of the interface between biological cell tissue and hard substrate materials using crossbeam technology. Microsc. Microanal. 11, 65–66 (2005)Google Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Center for Biomolecular Nanotechnologies @UNILE, IITArnesanoItaly
  2. 2.NNL-National Nanotechnology LaboratoryCNR-NANO, Università del SalentoLecceItaly

Personalised recommendations