Beam Deceleration for Block-Face Scanning Electron Microscopy of Embedded Biological Tissue

Micron. 2012 Apr;43(5):612-20. doi: 10.1016/j.micron.2011.11.001. Epub 2011 Nov 9.

Abstract

The beam deceleration (BD) method for scanning electron microscopes (SEM) also referred to as "retarding" was applied to back-scattered electron (BSE) imaging of the flat block face of a resin embedded biological specimen under low accelerating voltage and low beam current conditions. BSE imaging was performed with 0-4 kV of BD on en bloc stained rat hepatocyte. BD drastically enhanced the compositional contrast of the specimen and also improved the resolution at low landing energy levels (1.5-3 keV) and a low beam current (10 pA). These effects also functioned in long working distance observation, however, stage tilting caused uncorrectable astigmatism in BD observation. Stage tilting is mechanically required for a FIB/SEM, so we designed a novel specimen holder to minimize the unfavorable tilting effect. The FIB/SEM 3D reconstruction using the new holder showed a reasonable contrast and resolution high enough to analyze individual cell organelles and also the mitochondrial cristae structures (~5 nm) of the hepatocyte. These results indicate the advantages of BD for block face imaging of biological materials such as cells and tissues under low-voltage and low beam current conditions.

MeSH terms

  • Animals
  • Hepatocytes / ultrastructure*
  • Histological Techniques / instrumentation
  • Histological Techniques / methods
  • Imaging, Three-Dimensional / instrumentation*
  • Imaging, Three-Dimensional / methods*
  • Microscopy, Electron, Scanning / instrumentation*
  • Microscopy, Electron, Scanning / methods*
  • Rats
  • Rats, Wistar