The three-dimensional (3D) analysis of anatomical ultrastructures is extremely important in most fields of biological research. Although it is very difficult to perform 3D image analysis on exact serial sets of ultrathin sections, 3D reconstruction from serial ultrathin sections can generally be used to obtain 3D information. However, this technique can only be applied to small areas of a specimen because of technical and physical difficulties. We used ultrahigh voltage electron microscopy (UHVEM) to overcome these difficulties and to study the chemical neuroanatomy of 3D ultrastructures. This methodology, which links UHVEM and light microscopy, is a useful and powerful tool for studying molecular and/or chemical neuroanatomy at the ultrastructural level. 1. Introduction The three-dimensional (3D) analysis of anatomical ultrastructures is extremely important in most fields of biological research. However, it is considerably difficult to perform a 3D image analysis of exact serial sets of ultrathin sections. Although 3D reconstruction from ultrathin sections (~100？nm thickness) has been generally used to obtain 3D information, this technique is applicable only for small specimen areas because of the technical and physical difficulties under the transmission electron microscopy, restricted to approximately 1？mm2 area. On the other hand, due to tremendous development of various techniques in molecular biology (e.g., green fluorescent proteins and their color variants), as well as the development of live imaging techniques, the structure of biological molecules and their functional changes are calculated and visualized in 3D at subnanometer resolution [1, 2]. With the aid of confocal laser scanning microscopy, it is now possible to image and quantify the 3D organization of these cell processes; however, the detailed morphology of the complicated terminal processes of these cells remains obscure because of the insufficient spatial resolution of light microscopy and visualization methods that depend on fluorescence [3–6]. In addition, unstained domains are very difficult to recognize [3]. In contrast, conventional transmission electron microscopy provides extremely detailed and fine structural information, but the images obtained are mostly 2D due to the physical properties of this imaging technique (use of ultrathin sections). Consequently, it is too difficult to relate electron micrographs to the 3D structures of cells. The high penetration power of electrons at an ultrahigh accelerating voltage enables the examination of thick sections of biological
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