Proceedings of the Anatomical Society of Great Britain and Ireland.

Abstract

Iodine has been used as a contrast agent in prior applications in x-ray and electron microscopic imaging. Here we used methacrylate copolymers with iodine covalently linked to their backbone structure as embedding medium. These materials were prepared from methylmethacrylate (MMA) and 2-[4’-iodobenzoyl]-oxo-ethylmethacrylate (4-IEMA) (Kruft et al., J. Biomed. Mater. Res. 28, 1994). Mouse and horse bone samples fixed in NFS or 70% ethanol were embedded using a molar ratio of 4-IEMA:MMA of 1:3.6 giving an empirical formula of C $" H %".) O "".# I. Block surfaces were finished by diamond ultramilling and carbon coated. Digital backscattered electron (BSE) images were recorded using a Zeiss DSM962 SEM. Images at nominal 500X and 2048* pixel resolution at 20 kV showed no evident beam damage. At 30 kV, increasing the BSE yield and thereby the working distance and field of view, we could image wide fields (e.g. 15 mm) in a single scan. Image contrasts in cells and soft tissues reflect the inverse of the mass concentration of organic solids retained after the embedding protocol : proteins, DNA, RNA are retained; lipids, being soluble in ethanol and methyl methacrylate monomer are lost, but this is exactly as in conventional histological procedures. Advantages over the latter concern our ability to prepare very flat and undeformed samples. Our material is a solid block, with one or more surfaces flattened to ! 0.1 μm roughness. At 20 kV, the information depth is of the order of 0.5 μm. It is not possible to generate extensive physical sections that are so thin. Furthermore, sections of mixtures of tissues with varying degrees of hardness are deformed and do not lie within a conventional optical section plane. Thus the new method may have significant applications in discreet problem areas.