High-Resolution, Low Voltage, Field-Emission Scanning Electron Microscopy (HRLVFESEM) Applications for Cell Biology and Specimen Preparation Protocols

  • Heide Schatten


High resolution, low-voltage, field-emission scanning electronmicroscopy (HRLVFESEM), coupled with newly-developed specimen preparation protocols has permitted novel and powerful applications for cell biology, as it allows detailed insights into small biological features due to the minimal coating requirements. It allows resolution at levels that previously could only be achieved with transmission electron microscopy (TEM) and it has made it possible to generate three-dimensional images of structures as well as interactions with macromolecular complexes without confusion of structural overlap which allows clear interpretation, particularly when stereo-imaging is applied. In this chapter several examples are presented using three specific preparation techniques that can be applied to a variety of different specimens with specimen-specific modifications. The three separate sections in this chapter are 1) Visualization of sub-membranous cytoskeletal features using cytoskeleton stabilization and membrane extraction protocols. Two examples of different specimens in this section are (1.1) subpellicular cytoskeletal structures of the apicomplexan parasite Toxoplasma gondii, and (1.2) submembraneous actin cytoskeleton in osteocytes; 2) Visualization of whole mounts and isolated cell structures. The examples presented in this section include (2.1) Visualization of isolated nuclear envelope; (2.2) visualization of isolated mitotic spindles; (2.3) visualization of centrosomes; and 3) Visualization of resin-extracted de-embedded thick sections. De-embedding of thick-sectioned biological material is a unique approach to examine the interior of cells and tissue. Serial sections can be obtained and the entire cell or tissue can be evaluated by 3-D reconstructions of thick-sectioned material. In addition, the area of interest can be tilted and viewed from various angles for accurate interpretation. The power of this unique method is demonstrated in three applications focused on: (3.1) Nuclear pore complexes (Ris and Malecki, 1993); (3.2) Muscle fibers (Ris and Malecki, 1993); (3.3) Toxoplasma parasite internal structures. All methods presented in this chapter provide unique approaches to visualize and analyze delicate biological structure.


Tannic Acid Mitotic Spindle Stereo Image Nuclear Pore Complex Toxoplasma Gondii 
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© Springer Science+Business Media, LLC 2008

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  • Heide Schatten

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