Three-dimensional visualization of Golgi-stained neurons by a projection X-ray microscope converted from a scanning electron microscope.

Trying to obtain the three-dimensional images of neuronal elements, Golgi-stained thick sections were examined by a projection X-ray microscope converted from a scanning electron microscope. An attachment unit for the projection X-ray microscope consists of a target holder, specimen stage and film cassette. Two kinds of target material, titanium and gold, were tested. The system was operated at 10 to 30 kV. The electron beam into the target was about 20 nA with the use of a pointed cathode. The stereopair of X-ray micrographs were taken by tilting the specimens. Quality and contrast of the X-ray micrograph taken with a titanium target was better than that with a gold target. The spatial resolution was estimated about 1 to 2 micrometers for 100 to 350 micrometers thick sections with a titanium target. Compared with the light microscope, strong penetrating ability of X-ray permitted to visualize the internal structures of optically opaque materials. Further, almost all objects were in focus in X-ray micrographs even for relatively thick sections (100 to 350 micrometers). The three-dimensional morphology of neurons and glial cells was demonstrated by a stereopair of X-ray micrographs.