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Targeted Studies Using Serial Block Face and Focused Ion Beam Scan Electron Microscopy
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Imaging three-dimensional tissue architectures by focused ion beam scanning electron microscopy.

Andrew J Bushby1, Kenneth M Y P'ng, Robert D Young

  • 1The NanoVision Centre, School of Engineering and Materials Science, Queen Mary, University of London, UK. a.j.bushby@qmul.ac.uk

Nature Protocols
|June 4, 2011
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Summary

This protocol details volume electron microscopy (VEM) or focused ion beam scanning electron microscopy (FIB/SEM) for 3D biological tissue imaging. It reveals complex microanatomy with subcellular resolution, aiding fields like pathology and developmental biology.

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Area of Science:

  • Biological imaging
  • Microscopy techniques
  • Electron microscopy

Background:

  • 3D imaging of biological tissues is crucial for understanding complex microanatomy.
  • Existing techniques may lack the resolution or volume capacity for detailed structural analysis.
  • Subcellular resolution in large tissue volumes is needed across various biological disciplines.

Purpose of the Study:

  • To describe a protocol for volume electron microscopy (VEM) using focused ion beam scanning electron microscopy (FIB/SEM).
  • To enable high-resolution 3D imaging of biological tissues.
  • To demonstrate the application of FIB/SEM for revealing complex microstructures.

Main Methods:

  • Utilized a scanning electron microscope with a focused gallium ion beam for sequential milling.
  • Employed a backscattered electron detector to image milled surfaces, generating serial images.
  • Developed methods for tissue processing, contrast enhancement (osmium tetroxide/potassium ferricyanide), and data acquisition over ~120 hours.

Main Results:

  • Achieved structural information over volumes of tens of thousands of cubic micrometers.
  • Revealed complex microanatomy with subcellular resolution in biological tissues.
  • Illustrated the technique's utility in analyzing collagen fibril organization in developing chick cornea.

Conclusions:

  • Volume electron microscopy (FIB/SEM) provides a powerful method for high-resolution 3D imaging of biological tissues.
  • The protocol enables visualization of complex microstructures essential for tissue function.
  • This technique has broad applicability in pathology, developmental biology, anatomy, and regenerative medicine.