Beam deceleration for block-face scanning electron microscopy of embedded biological tissue

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.

► Beam deceleration (BD) applied to a block face SEM imaging (BFI) of embedded cell. ► BD drastically enhanced the contrast and resolution (∼5 nm) of the BFI of a cell. ► Effect remarkable at low accelerate voltage (1.5 kV) and low beam current (10 pA). ► Newly developed device allows 3D imaging using FIB/SEM with BD. ► BD improved 3D reconstruction allows mitochondrial cristae structural analysis.