Table 1. Comparison of the new tSEM versus TEM systems.
| tSEM | TEM | ||
| Platform | FE-SEMa | TEM | |
| Specimen holder | multiple grids at a time | usually one grid at a time (some special holders) | |
| Illumination | rastered probe | entire image field | |
| Accelerating voltage | 28 kV | 120 kV | |
| Detection method | transmitted electron detector | CCD camera | |
| Automation | Routine: Stage translation, image optimization (focus, brightness, etc.), image acquisition, and mosaic. | Specialized: Some modules for focus and astigmatism compensation, some mosaic functions.b | |
| Usual operator involvement | Identify center of image fields & set scan rotation on each section | Repeated specimen exchange & stage translation, physical specimen rotation, image optimization & acquisition | |
| Relative cost of instrument: | ∼Half that of manual TEM | ||
| (a) | Optimal pixel size (in x–y) for analysis | 2 nm | 2 nm |
| (b) | Section thickness | 45 nm | 45 nm |
| Voxel size ( = a×a×b) | 2 nm×2 nm×45 nm | 2 nm×2 nm×45 nm | |
| (c) | Typical number of serial sections | 200 | 200 |
| (d) | Maximum single field dimensions | 32,768 pixels per side | 4,080 pixels per side |
| (e) | Maximum single image field dimensions ( = a×d) | 65.54 µm per side | 8.160 µm per side |
| (f) | Maximum single image field area ( = e×e) | 4,295 µm2 | 66.59 µm2 |
| (g) | Volume of imaged tissue ( = b×c×f) | 38,660 µm3 | 599.3 µm3 |
| Total number of voxels ( = c×d×d) | 2.147×1011 voxels | 3.329×109 voxels | |
| (h) | Operator time to acquire single field image over 200 sections | ∼4 hr (plus additional ∼74 hr for unattended image acquisition).c | ∼40 hr. |
| Volume of imaged tissue per operator time ( = g/h) | 9664 µm3/hr | 14.98 µm3/hr |
a
At the time of this writing, Zeiss is the sole source of the FE-SEM system designed for high-resolution large-field transmission imaging of biological samples as described here.
b
TEM can be retrofitted to acquire mosaic images automatically with open source software such as Leginon (National Resource for Automated Molecular Microscopy, The Scripps Research Institute; http://www.leginon.org/) and SerialEM (The Boulder Lab for 3D Electron Microscopy, University of Colorado; http://bio3d.colorado.edu/SerialEM/).
c
Note that the operator time for tSEM remains constant for the same number of serial sections, even when the volume size is increased by montaging.