Scanning Electron MicroscopyWith the increasing number of advanced imaging tools available, the utility of conventional imaging techniques is often overlooked. In fact, the ability to visualize structures with the high resolution achieved by using electron microscopes provides the foundation for developing valid conclusions about functional relationships. Despite advances in other types of light (LM), atomic force microscopy (AFM), and electron microscopy (EM), scanning electron microscopy (SEM) remains distinct in its ability to examine dimensional topography and distribution of exposed features. The ultimate resolution achieved is controlled both by optimizing specimen preparation and instrumental parameters.Preservation of biological structures in a manner that prevents decay under the high vacuum necessary for a mean free path of travel for the electron beam is a primary concern. Biological specimens are generally composed of non-conductive, thermally sensitive, fragile material which if not stabilized results in specimen damage and imaging artifacts. Specimens can be prepared through chemical and physical (i.e. cryo-preservation) methods or both. Although cryo-preservation may be the preferred method for optimal near native
NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript state preservation, it requires expensive instrumentation, advanced skills, and is not necessary to address many scientific questions. In general, preparation of specimens for examination by SEM requires stabilization of structures as described in Basic Protocol 1, then dehydration of specimens as described in Basic Protocol 2, and finally, ensuring adequate conductivity as discussed in Basic Protocol 3, or their respective alternatives.Specifically, Basic Protocol 1 provides some protocols and tips for chemical preservation of animal tissues and cells, bacteria, viruses, and macromolecular specimens. Basic Protocol 2 provides options for specimen mounts and critical point or chemical drying, and Basic Protocol 3 addresses the rationale for metal coating and alternatives. Additional techniques for immune-labeling strategies with special considerations for correlative techniques are outlined in Basic Protocol 4, and "affordable" specimen fracture is discussed in Basic Protocol 5. Basic Protocol 6 describes stereo pair and anaglyph generation to produce 3-dimensional (3-d) images. The Commentary section discusses basic theory and application for both preparative and imaging techniques of biological specimens for SEM and offers a troubleshooting guide for common problems.There is no one method or condition that assures successful preparation and imaging for all biological specimens, thus the user should be willing to experiment with preparative and imaging techniques and technologies to achieve the desired results. The content of this Unit is not meant to be exhaustive, but rather offers a starting point for biological specimen preparation and imaging by SEM.
Safety ConsiderationsPreparation of biological specimens f...