High Voltage Electron Microscopy for Thick Sections of Biological Materials Combined with Molecular Tracers

Abstract

One of the advantages of high voltage electron microscopy (HVEM) is the great penetration of electron beam, which permits the examination of thicker sections of resin-embedded biological materials. However, when stained thick sections are examined under the HVEM, all structural details within the whole thickness are brought into focus as superimposed image. Hence, certain structures are not easily observable on such an image, even on the stereo view obtained by specimen tilting technique. To overcome this difficulty, molecular tracers are introduced to the HVEM in an attempt to visualize certain structures selectively.Mouse kidney and cardiac muscle were examined using ruthenium red and peroxidase as molecular tracers. Thick Epon sections ranging from 0.5 to 3 μ were observed in a JOEL JEM-1000 or a Hitachi HU-1000 HVEM, with an accelerating voltage of 1000 KV. Pictures were taken routinely as stereo pairs using a tilting stage device with ±10° or ±8°.