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What is the difference between immuno-EM, thin-section TEM, and ET?

What is the difference between immuno-EM, thin-section TEM, and ET?

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  • George Komis · Palacký University of Olomouc
    for thin sectioning TEM with unspecific contrasting, fixation and resin embedding are more robust, preserving structure very well but in the expence of antigenicity. For immunoTEM, fixation regimes are milder, while resins used are normally hydrophilic, do not copolymerize with biopolymers and polymerize at low temperatures necessary to preserve antigenicity. Depending on what your needs are you may google TEM, or immunogold and come up with thousands of protocols that will comply with the material you are working with.
  • Isabel Cristina Rodriguez Rodriguez · University of Antioquia
    Thanks
  • Wolfgang Muss · Paracelsus Medical University Salzburg
    Dear Isabel,
    the answer might be somehow broadened, if I know / one knows what you definitively mean with “ET”. Perhaps you can reply to this.
    In addition to George Komis’s answer I would like to explain what “thin section TEM” might mean too.
    Unfortunately we don’t know the background of your question.
    So, for <Immuno-EM> my definition is (very briefly) to localize an antigenic site enclosed in tissular structure (as contained in usually an ultrathin section, which is around 60-70 nm thick) by using specific protocols for immunolabeling (“staining”) such sections with “labeled” antibodies to the respective antigen. There are two main procedures to do so: “pre-embedding”- or “postembedding”-labeling techniques (the former will label antigens in tissular structures prior to (fixing) –at least dehydration of tissue blocks, the latter after irreversible fixation, dehydration, embedding in resin, performed ON the ultrathin sections, therefore needing also special techniques to maintain the antigenicity, as George said. For the latter there also exist special techniques using other resins than used for classical, routine TEM, mostly “acrylic” resins (which also can be polymerized at lower temperatures than usual). Very modern TEM’s or TEM-applications are now capable to not only display usual structural (and differential) e-dense contrast (which has been applied prior to examination in the EM by heavy metal salts like [most common] uranyl-acetate, and /or Lead citrate or other stains like Bi-nitrate, Pt-blue, etc.) but also can examined for fluorescence staining applied during processing of tissues (e.g. FEI-company and others) .
    “Thin section <TEM>” should cover the “classical approach” where tissues after fixation, dehydration etc. are embedded in resin (usually epoxy-/epoxide type). Such resins are polymerized at higher temperatures (up to 60-80°C, therefore merely unsuited for Immuno-(T)EM-approaches) and the resulting ultrathin sections have a thickness of approx. 40-70 nm depending on skills and study purpose, but for special use such have to be either thinner (25-30 nm, e.g. for EELS) or (depending on the possibility to gain higher accelerating voltage = 100 KV, 200 kV etc. of the TEM used) one might also use thicker sections (up to 100-150 nm, & 0.25 µm or more). Such thicknesses have been used for "T"-EM (= 2D/3D-electron tomography) whereby by tilting specimens in the e-beam and software programs (digital microscopes mandatory) one can produce 3D-reconstructions and/or 3D-animations from such thick slices.
    In the American and sometimes also English literature the term “thick section” also stands for so called “semithin sections” which usually are produced for locating specific areas of a tissue block, to be examined by ( “normal” ) Light microscopy prior to TEM examination.
    If I am right with my assumption that with the abbreviation “ET” you meant “e-tomography” I would like to point you first to the Wikipedia-article @
    < en.w...
    [more]
  • Kristine Atkinson · BioÄventyr Technologies
    Immuno TEM is done with different reagents and embedding protocols in order to preserve antigenicity for an ELISA performed on ultrathin sections. The operator should first be highly proficient in standard TEM, as the added difficulties require understanding what step was harmful, such as lack of preservation at the fixation step, or leaching of components during dehydration.
    A standard embedment uses the resin Lowicryl, which requires ultraviolet-induced polymerization in a freezer: imagine making tiny capsules available for UV exposure in a freezer for several days; I designed a mesh holder for the purpose. It is a brittle polymer, hard to section (I found fresh glass knives superior to diamond), and not happy under the electron beam, with some movement affecting long micrograph exposures. Fixation can tricky, and visualization without osmium sometimes depends on good focus and trusting the photograph will capture what was there.
    But when all works well, the results are spectacular: you can easily do 3-D via stereo pairs for localization of the ultrafine gold particles at the supermolecular level, and we were able to show that an immunoglobulin was not transported free in the cytoplasm, but was actually bound to microvesicles. Well worth the effort.
  • Maria-Julieta Gonzalez · University of Chile
    ETM= Electron transmission microscopy= Microscopia electronica de transmisión.
    Cuando tú deseas hacer una observación con este microscopio, debes utilizar cortes de tejidos muy delgados que han sido previamente fijados. La fijación del tejido va a depender cual es tu objetivo. Luego estos tejidos son deshidratados en concentraciones crecientes de etanol y luego incluidos dentro de una resina. Recien ahí podras obtener los cortes finos. Si tu objetivo es hacer la identificación de un antigeno de tu celula o tejido para luego identificar con un anticuerpo, debes fijar el tejido e incluirlo en componentes que te permitan mantener la antigicidad de tu molecula que andas buscando. No hay protocolos convencionales que te sirvan para cualquier proteína, por ejemplo. Una vez obtenido el corte delgado, que se hace en un ultramicrotomo utilizando cuchillas de diamante, debes incubar el corte o sección con el primer anticuerpo y luego con un segundo anticuerpo que puede estar unido a particulas de oro o a una enzima. Si esta unido a oro tu observaras al microscopio electronico de transmisión unas esferas de X nm segun lo que hayas usado. Si usaste un Ab secundario unido a peroxidasa, por ejemplo, deberas incubar en una solución que tendra el sustrato necesario para que actue la enzima, luegoese precipitado lo observarás como un aterial difuso, no particulado en tu tejido o celula.
    ojala que esta información te sirva, porque tu pregunta esta confusa
    cariños
    desde Chile
  • Carmen Mannella · Wadsworth Center, NYS Department of Health
    I read this thread fast. ET usually refers to Electron Tomography, which can be applied to conventionally prepared plastic sections (thicker than depending on accelerating voltage of your TEM) or to cryo (frozen-hydrated) specimens, if your TEM can operate in an appropriate low dose mode with sensitive CCD and (preferably) automated image recording. ET involves recording 100 or more images from a specimen serially rotated in 1-2 degree increments around (usually) one or two axes at angles up to +/1 60-10 degrees. Software is available for transforming the tilted views into a 3D reconstruction. I (and I'm sure others) can provide more information about options if interested. By the way, ET can be combined with immuno-EM, for example, by pre-embedding immuno-gold labeling of plastic specimens or of frozen-hydrated suspensions of viruses, membranes etc (in latter case, can see antibodies directly, without gold, or can use probes line nano-gold). Lots of options.
  • Carmen Mannella · Wadsworth Center, NYS Department of Health
    My response should have read: at tilt angles up to +/- 60-70 (not 10) degrees (my apologies -- want to get a good researchgate score for this!). You can tell this means your TEM needs a good, computer controlled goniometer. Cryo stage would be needed for frozen-hydrated specimens....A lot of these practical issues are discussed in McEwen et al Methods in Cell Biology 89 (2009) 129

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