STEM tomography of high-pressure frozen and freeze-substituted cells: a comparison of image stacks obtained at 200 kV or 300 kV
Scanning transmission electron microscopic (STEM) tomography of high-pressure frozen, freeze-substituted semi-thin sections is one of multiple approaches for three-dimensional recording and visualization of electron microscopic samples. Compared to regular TEM tomography thicker sample sections can be investigated since chromatic aberration due to inelastic scattering is not a limit. The method is ideal to investigate subcellular compartments or organelles such as synapses, mitochondria, or microtubule arrangements. STEM tomography fills the gap between single-particle electron cryo-tomography, and methods that allow investigations of large volumes, such as serial block-face SEM and FIB-SEM. In this article, we discuss technical challenges of the approach and show some applications in cell biology. It is ideal to use a 300-kV electron microscope with a very small convergence angle of the primary beam (“parallel” beam). These instruments are expensive and tomography is rather time consuming, and therefore, access to such a high-end microscope might be difficult. In this article, we demonstrate examples of successful STEM tomography in biology using a more standard 200-kV microscope equipped with a field emission tip.
KeywordsSTEM tomography Cell biology Synapse Electron microscopy High pressure freezing Freeze substitution
We thank Giada Frascaroli for the M2 macrophage samples. We thank Renate Kunz for preparing the up to 1-µm-thick sections and mounting them on the copper grids and Reinhard Weih for keeping all our equipment alive. We thank Clarissa Read (formerly known as Clarissa Villinger) and Reinhard Rachel, Regensburg, for helpful discussions. We thank Ingo Daberkow from Tietz Systems GmbH for his abundance of patience at the phone, answering our amateurish questions, and for always getting our STEM system back to work. We finally thank David Mastronarde and his team for developing the IMOD software and making it freely available for everybody and for the counseling via the IMOD list.
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