Comparison of 3D STEM with TEM Tomography for Biology

3D STEM is a 3D technique for the imaging of whole assemblies, as is tilt-series TEM, in contrast to the averaging techniques using diffraction from crystals or multiple images ofidentical objects. The resolution of the tomogram obtained in a tilt series (both on cryo and embedded or stained) is typically 5-10 nm in xyz [36-40]. From our calculations it follows that 3D STEM with the present microscopes already has approximately the same resolution (4 x 4 x 7 nm) on a conventional thin section. Improvement of the resolution is expected to be possible with a dedicated deconvolution procedure and with a new generation of aberration corrected microscopes.

The second advantage will be provided by the speed of the imaging technique. A focal series is readily recorded in 5 min, without need for realignment of adjustment of the microscope. TEM tomography, even automated [126,127], is still a delicate technique where manual alignment on markers added on the sides of the sample is often required. The sample does not have to be tilted such that larger and thicker samples can be imaged without suffering from beam blurring and focusing issues. Moreover, the absence of a tilt series reduces the drift alignment and magnification correction. 3D STEM could also be acquired at several tilt angles, combining the advantages of both techniques. The data acquisition is not limited to a data set representing a cubic volume, but 3D STEM can in principle acquire a data set of any shape. For example, a long and thin object, such as an axon, could readily be captured with only a minimum number of surrounding pixels using selected scanning of the electron beam. As a result, objects with elongated shapes would be captured as a whole within a data set of reduced size.

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