, Volume 246, Issue 1–4, pp 41–48 | Cite as

Focused ion beam (FIB)/scanning electron microscopy (SEM) in tissue structural research

  • Vladka Lešer
  • Marziale Milani
  • Francesco Tatti
  • Živa Pipan Tkalec
  • Jasna Štrus
  • Damjana Drobne
Original Article


The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.


Histopathology Hepatopancreas Terrestrial isopods Crustacea EDX analyses 



Focused ion beam


Scanning electron microscopy


Transmission electron microscopy


Energy dispersive X-ray


Back-scattered electrons



We thank FEI Italy for the access to Strata DB235 M at the University of Modena e Reggio Emilia, Italy. This work was supported by the Slovenian Ministry of Higher Education, Science and Technology (grants J1-6473 and 3311-03-831253), World Federation of Scientists, and the Slovenian Science Foundation (WFS National Scholarship SZF-V.Lešer/WFS-01/2007). We thank Bill Millne for the English editing.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Bozzola JJ, Russell LD (1998) Electron microscopy: principles and techniques for biologists. Jones and Bartlett Publishers, SudburyGoogle Scholar
  2. Chaiwan S, Hoffman M, Munroe P, Stiefel U (2002) Investigation of sub-surface damage during sliding wear of alumina using focused ion-beam milling. Wear 252:531–539CrossRefGoogle Scholar
  3. Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLOS Biol 2:1900–1909CrossRefGoogle Scholar
  4. De Winter DAM, Schneijdenberg CTWM, Lebbink MN, Lich B, Verkleij AJ, Drury MR, Humbel BM (2009) J Microsc 233:372–383CrossRefPubMedGoogle Scholar
  5. Drobne D, Milani M, Ballerini M, Zrimec A, Berden Zrimec M, Tatti F, Drašlar K (2004) Focused ion beam for microscopy and in situ sample preparation: application on a crustacean digestive system. J Biomed Opt 9:1238–1243CrossRefPubMedGoogle Scholar
  6. Drobne D, Milani M, Lešer V, Tatti F (2007) Surface damage induced by FIB milling and imaging of biological samples is controllable. Microsc Res Tech 70:895–903CrossRefPubMedGoogle Scholar
  7. Drobne D, Milani M, Lešer V, Tatti F, Zrimec A, Žnidaršič N, Kostanjšek R, Štrus J (2008) Imaging of intracellular spherical lamellar structures and tissue gross morphology by a focused ion beam/scanning electron microscope (FIB/SEM). Ultramicroscopy 7:663–670CrossRefGoogle Scholar
  8. Hagedorn M, Ziegler A (2002) Analysis of Ca2+ uptake into the smooth endoplasmic reticulum of permeabilised sternal epithelial cells during the moulting cycle of the terrestrial isopod Porcellio scaber. J Exp Biol 205:1935–1942PubMedGoogle Scholar
  9. Hames CAC, Hopkin SP (1991) A daily cycle of apocrine secretion by the B cells in the hepatopancreas of terrestrial isopods. Can J Zool 69:1931–1937CrossRefGoogle Scholar
  10. Haswell R, McComb DW, Smith W (2003) Preparation of site-specific cross-sections of heterogeneous catalyst prepared for focused ion beam milling. J Microsc 211:161–166CrossRefPubMedGoogle Scholar
  11. Hayles MF, Stokes DJ, Phifer D, Findlay KC (2007) A technique for improved focused ion beam milling of cryo-prepared life science specimens. J Mirosc 226:263–269CrossRefGoogle Scholar
  12. Hekking LHP, Lebbink MN, De Winter DAM, Schneijdenberg CTWM, Brand CM, Humbel BM, Verkleij AJ, Post JA (2009) Focused ion beam-scanning electron microscope: exploring large volumes of atherosclerotic tissue. J Microsc 235:336–347CrossRefPubMedGoogle Scholar
  13. Heymann JAW, Hayles M, Gestmann I, Giannuzzi LA, Lich B, Subramaniam S (2006) Site-specific 3D imaging of cells and tissues with a dual beam microscope. J Struct Biol 155:63–73CrossRefPubMedGoogle Scholar
  14. Heymann JAW, Shi D, Kim S, Bliss D, Milne JLS, Subramaniam S (2009) 3D Imaging of mammalian cells with ion-abrasion scanning electron microscopy. J Struc Biol 166:1–7CrossRefGoogle Scholar
  15. Hopkin SP, Martin MH (1982) The distribution of zinc, cadmium, lead and copper within the hepatopancreas of a woodlouce. Tissue Cell 14:703–715CrossRefPubMedGoogle Scholar
  16. Inkson BJ, Mulvihillb M, Mobus G (2001) 3D determination of grain shape in a FeAl-based nanocomposite by 3D FIB tomography. Scripta Mater 45:753–758CrossRefGoogle Scholar
  17. Knott G, Marchman H, Wall D, Lich B (2008) Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J Neurosci 19:2959–2964CrossRefGoogle Scholar
  18. Lešer V, Drobne D, Pipan Ž, Milani M, Tatti F (2009) Comparison of different preparation methods of biological samples for FIB milling and SEM investigation. J Microsc 233:309–319CrossRefPubMedGoogle Scholar
  19. Mackenzie RAD, Smith GDW (1990) Focused ion beam: a bibliography. Nanotechnology 1:163–201CrossRefGoogle Scholar
  20. Morgan AJ, Winters C, Yarwood A, Wilkinson N (1995) In vivo metal substitutions in metal sequestering subcellular compartments: X-ray mapping in cryosections. Scan Microsc 9:1041–1060Google Scholar
  21. Perrey CR, Carter CB, Michael JR, Kotula PG, Stach EA, Radmilovic VR (2004) Using the FIB to characterize nanoparticle materials. J Micsosc 214:222–236CrossRefGoogle Scholar
  22. Phaneuf MW (1999) Applications of focused ion beam microscopy to materials science specimens. Micron 30:277–288CrossRefGoogle Scholar
  23. Sivel VGM, Van den Brand J, Wang WR, Mohdadi H, Tichelaar FD, Alkemade PFA, Zandbergen HW (2004) Application of the dual-beam FIB/SEM to metals research. J Microsc 214:237–245CrossRefPubMedGoogle Scholar
  24. Wägele JW (1992) Isopoda. In: Harison FW, Humes AG (eds) Microscopic anatomy of invertebrates. Crustacea, vol. 9. Wiley-Liss, New York, pp 529–617Google Scholar
  25. Young RJ (1993) Micro-machining using focused ion beam. Vacuum 44:353–356CrossRefGoogle Scholar
  26. Young RJ, Moore MV (2005) Dual-beam (FIB-SEM) systems. In: Gianuzzi LA, Stevie FA (eds) Introduction to focused ion beams. Springer, New York, pp 247–268CrossRefGoogle Scholar
  27. Young RJ, Dingle T, Robinson K, Pugh PJA (1993) An application of scanned focused ion-beam milling to studies on the internal morphology of small arthropods. J Microsc 172:81–88Google Scholar
  28. Žnidaršič N, Štrus J, Drobne D (2003) Ultrastructural alterations of the hepatopancreas in Porcellio scaber under stress. Environ Toxicol Pharmacol 13:161–174CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Vladka Lešer
    • 1
  • Marziale Milani
    • 2
  • Francesco Tatti
    • 3
  • Živa Pipan Tkalec
    • 1
  • Jasna Štrus
    • 1
  • Damjana Drobne
    • 1
  1. 1.Department of Biology, Biotechnical FacultyUniversity of LjubljanaLjubljanaSlovenia
  2. 2.Materials Science Department and Laboratory FIB/SEM “Bombay”University of Milano-BicoccaMilanItaly
  3. 3.FEI ItaliaMilanItaly

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