Abstract
Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.
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Acknowledgments
We thank Renate Kunz, Eberhard Schmid and Reinhard Weih for excellent technical assistance. This project was partially supported by the BMBF project NanoCombine. BM and CK acknowledge the support by FEI Co. (Eindhoven, Netherlands), the German Science Foundation (INST40/385-F1UG) and the Struktur- und Innovationsfonds Baden-Wuerttemberg.
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418_2012_1020_MOESM1_ESM.m4v
Video 1: FIB/SEM dataset of Fig. 2 according to Protocol A. A Volume of 25 μm × 19 μm × 6.5 μm is visible in this slice and view dataset. The ultrastructural features such as mitochondria or the Golgi apparatus are well preserved and well resolved (M4V 27,070 kb)
418_2012_1020_MOESM2_ESM.m4v
Video 2: FIB/SEM dataset of Fig. 4 according to Protocol B. A volume of about 2 μm × 1.5 μm × 3 μm is visible in this “slice and view” dataset, recorded at high magnification. Note the good visibility of fine structural details such as the two leaflets of the membrane bi-layer and ribosomes (M4V 9,710 kb)
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Villinger, C., Gregorius, H., Kranz, C. et al. FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells. Histochem Cell Biol 138, 549–556 (2012). https://doi.org/10.1007/s00418-012-1020-6
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DOI: https://doi.org/10.1007/s00418-012-1020-6