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Electron Microscopy Techniques FAQ

  1. What kinds of electron microscopy techniques are available?
  2. Are immuno-electron microscopy services available?
  3. How should I bring samples to the Facility for electron microscopy? If I bring cells, how should they be delivered?
  4. Can I bring formalin-fixed or paraffin-embedded samples for TEM?
  5. If I don't need immuno-EM, how long does it take to prepare samples for viewing on the electron microscope?
  6. Why does the sample size (tissue) have to be so small?

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  1. What kinds of electron microscopy techniques are available?

    We have four electron microscopes: two scanning electron microscopes (SEM) and two transmission electron microscopes (TEM).

    SEMs are great for looking at the microscopic surfaces of objects, ranging from biomaterial scaffolds, cells, tissues, and bone. In particular, the LEO Field Emission SEM can reveal protein complexes on the surfaces of cells. In addition, we have developed selective extraction methods so that subcellular structure (cytoskeleton) is revealed using SEM of whole cells after plasma membrane removal.

    TEMs are great for thin sections within tissues to reveal subcellular structures such as membranes. The details that TEM can reveal are exquisitely fine, almost molecular in spatial resolution. Immuno-electron microscopy services are available, too. In this technique, primary antibodies are conjugated to colloidal gold particles and used to decorate thin sections; they appear as dark spots in the TEM. In principle, multiple antibodies can be conjugated to different sized gold particles, but the specificity of antibodies and of conjugation are very demanding, requiring many control experiments.

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  2. Are immuno-electron microscopy services available?

    Yes, we will perform immuno-electron microscopy on your sample. In addition to providing the sample, you must provide the primary antibody for your study. Just as for any antibody study, the quality (specificity, affinity, and purity) of your antibody is critical for success. For immuno-electron microscopy, we caution you that Western blots are not a sufficient test of antibody quality. Westerns typically immobilize denatured peptides as antigens, whereas fixed or live cells and tissues have natively folded proteins (and protein complexes). Not only do antibodies lose apparent specificity when probing native rather than denatured peptides, protein complexes and cellular structures may prevent accessibility to antigenic epitopes. Please see the FAQ for immuno-electron microscopy for a more thorough discussion of the pre-requisites for successful immuno-electron microscopy.

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  3. How should I bring samples to the Facility for electron microscopy? If I bring cells, how should they be delivered?

    As long as there are no chemical or biological hazards (e.g. toxins, parasites, viruses), you can bring living samples to the Facility for processing services. However, you must arrange a delivery time in advance, so that we are ready to process your sample immediately upon receipt. In fact, we recommend a full consultation prior to deliver. We can discuss whether cells are delivered in carbonate-buffered growth media (we have a tissue culture incubator) or phosphate buffered media, temperature sensitivity of sample during transport, and any other relevant issues.

    Alternatively, you can also fix samples in your own lab. Please consult with us first. We have a wealth of protocols and experience, and may be able to guide you by anticipating challenges and suggesting customizations for your sample. Of course, use only electron-microscopy-grade (EM-grade) reagents, particularly glutaraldehyde. As for living samples, you should arrange a delivery time in advance so that we are ready to process your sample upon receipt.

    As with any procedure, the quality of your starting material affects the quality of imaging. Before you commit to extensive processing, please confirm that the morphology and behavior of your cells are correct. Indeed, you should consider using the optical microscope to monitor the fixation process. If we do the processing, you should be available to consult on the morphology during the processing. A simple glance at your sample on the microscope can avoid a lot of frustration.

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  4. Can I bring formalin-fixed or paraffin-embedded samples for TEM?

    No, given a choice, we do not recommend using formalin-fixation or paraffin-embedded tissues for electron microscopy. Typically, formalin fixation extracts too much material, thus affecting the fine structure of the sample. Harsh solvents (e.g. xylene) are required to remove paraffin, thus affecting ultrastructure, too. Although we have salvaged samples for some specialty circumstances, we can not guarantee the fidelity when attempting to process and image such samples.

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  5. If I don't need immuno-EM, how long does it take to prepare samples for viewing on the electron microscope?

    Upon receiving a live sample, a typical turn-around time is ~2 weeks for fixation, thin-sectioning, and mounting on EM grids for viewing. If rush-processing is requested and there are no conflicting commitments, we can sometimes accelerate specimen preparation to approximately 1 week. Please consult us for availability of rush-processing.

    For immuno-electron microscopy, refer to the FAQ for immuno-electron microscopy.

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  6. Why does the sample size (tissue) have to be so small?

    Samples (tissue slices) must be <3 mm3 for good morphology in electron microscopy. The main limitation is the speed and extent of penetration of fixatives (typically glutaraldehyde). If fixative doesn't penetrate sufficiently, then the specimen won't be sufficiently fixed for subsequent processing. In general, we will only section a 1 mm3 of that sample. Remember that TEM typically uses ~80 nm slices, so 1 mm provides ~12,500 slices — way too many to search for a rare feature. To "preview" or find interesting sections of the sample, we typically perform light microscopy of stained "semi-thin" sections (typically 0.5 µm). For rare features in the tissue, please be available to consult about areas to focus our efforts in thinner slices.

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