title: Beam deceleration for block-face scanning electron microscopy of embedded biological tissue.
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.
authors.affiliation: Division of Microscopic and Developmental Anatomy, Department of Anatomy, Kurume University School of Medicine, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. kohta@med.kurume-u.ac.jp
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ohta
authors.forename: Shoji
authors.initials: S
authors.lastname: Sadayama
authors.forename: Akinobu
authors.initials: A
authors.lastname: Togo
authors.forename: Ryuhei
authors.initials: R
authors.lastname: Higashi
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Tanoue
authors.forename: Kei-ichiro
authors.initials: K
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D022781:Hepatocytes / Q000648:ultrastructure*; D006652:Histological Techniques / Q000295:instrumentation / Q000379:methods; D021621:Imaging, Three-Dimensional / Q000295:instrumentation* / Q000379:methods*; D008855:Microscopy, Electron, Scanning / Q000295:instrumentation* / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar