Recent development of scanning electron microscopy (SEM) technique, by which images are taken from back-scattered electron (BSE) from flat block surface of resin-embedded biological specimens, enabled us to obtain high contrast images similar to the ones obtained by TEM. This imaging method facilitates not only a wide area of observation but also a 3D volume analysis such as a FIB/SEM tomography method. However, the BSE image obtained by a conventional SEM under general conditions does not have sufficient contrast and resolution to observe detailed structures of the cell. Here we present a “retarding” method that drastically enhances the quality of the BSE image even when obtained by the conventional SEM. The “retarding” method reduces the primary electron energy by the negative bias voltage between the specimen and the beam column, and it provides high resolution (4 nm) and high contrast images from the resin block surface when combined with optimal SEM settings. Furthermore, when the retarding method is applied to a FIB/SEM 3D reconstruction method, it can reconstruct a larger volume than is achievable by conventional electron tomography, and its high spatial resolution permits the visualization of 3D structures of the cell such as the membrane organization of organelles.
View full abstract