Same-section correlative light and electron microscopy

Abstract

Epoxy embedded biological material, sectioned for conventional, intermediate or high-voltage electron microscopy (EM), can be visualized within the section with good contrast and detail by phase-contrast or dark-field light microscopy (LM). The contrast of such material is not substantially influenced by the type of embedding resin or section support substrate. It is, however, influenced by the type of fixation (glutaraldehyde with and without osmium postfixation), by heavy metal (uranyl and lead) staining, and by the section thickness. The ability to examine the specimen with the LM, within a section cut for EM, allows each section to be rapidly screened for content prior to examination in the EM. We have found this approach to be particularly useful for studies requiring the ultrastructural examination of a selected area or structure which is large enough to be visualized with the LM but which comprises only a small volume of the embedded material (e.g., centrosomes and nuclei within oocytes; specific regions of large protists.)Same section LM-EM can also be extended to sections cut from cells stained prior to embedding for the immunofluorescent localization of antigens using fluorescein, rhodamine or Texas red-conjugated antibodies. Under these conditions the sections show high resolution patterns of antigen-specific fluorescence against a background void of autoflouorescence. The fidelity in the correlation between the distribution of immunofluorescently labeled antigens and the ultrastructure in the same section can be extended to structures as small as a single microtubule or just a few actin filaments . This approach of same section correlative fluorescence microscopy (FLM) and EM can eliminate the need, in many instances, to employ more complex procedures such as immunoferritin or immunogold for labeling antigens for ultrastructural detection.