STEM tomography for thick biological specimens.

Scanning transmission electron microscopy (STEM) tomography was applied to biological specimens such as yeast cells, HEK293 cells and primary culture neurons. These cells, which were embedded in a resin, were cut into 1-microm-thick sections. STEM tomography offers several important advantages including: (1) it is effective even for thick specimens, (2) 'dynamic focusing', (3) ease of using an annular dark field (ADF) mode and (4) linear contrasts. It has become evident that STEM tomography offers significant advantages for the observation of thick specimens. By employing STEM tomography, even a 1-microm-thick specimen (which is difficult to observe by conventional transmission electron microscopy (TEM)) was successfully analyzed in three dimensions. The specimen was tilted up to 73 degrees during data acquisition. At a large tilt angle, the specimen thicknesses increase dramatically. In order to observe such thick specimens, we introduced a special small condenser aperture that reduces the collection angle of the STEM probe. The specimen damage caused by the convergent electron beam was expected to be the most serious problem; however, the damage in STEM was actually smaller than that in TEM. In this study, the irradiation damage caused by TEM- and STEM-tomography in biological specimens was quantitatively compared.