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| Organelles and cellular structures |
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| TEM (Transmission Electron Microscopy) |
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| Beams of electrons focused by magnets through specimens that have been fixed and stained with heavy metals. Beam passes through unstained area and scatters depending on which area was stained. Can be used to view cells and organelles and evaluate some protein structures. |
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| SEM (Scanning electron microscopy) |
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| A beam of electron focuesed by a magnet hits a gold-coated substance which then reflects the electrons back to be captured and converted to an image; can be used to view 3D surface of biological specifmens. |
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| Phase Contrast Microscopy |
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| Not a lot of specific structure visibility; uses refraction to give a dark/light constrast to unstained tissue and cells. |
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| UV light excites a dye *chromaphore* molecule, which re-emits a longer and more specific wavelength. The sample is viewed through a filter on the eyepiece that only permits that specific wavelength to pass. Can be used to localize a specific protein in antibody labeled tissue. |
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| Unaltered/non-filtered visible light used to view tissue sections on glass slides. This is the most commonly used form of light microscopy for histology and pathology. |
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| Tissues are frozen in gelatin and sections are generated with a specialized microtome called a cryostat. Quick but results in poor resolution. |
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| Tissues are embedded in wax prior to sectioning; gives great resolution because fixation has fixed the molecule, the tissue has been cleared and paraffin fills the spaces where fat once resided. The sections are 5-10 micrometers thick. |
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| A buffered formaldehyde (formalin--a derivative) preservative fixes molecules and inactivates any enzymatic activity. |
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| Does not allow for removal of lipids or fixation of any sort; allows us to view enzymatic activity. Can be rapidly processed but the resolution is poor and cellular shapes cannot be effectively observed. |
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| Paraffin sections: pros and cons |
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| Can produce amazing images; however substances are hard to detect and process is slow-going. |
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| Hematoxylin and Eosin (H&E) |
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| acid/base reactions allow for uptake of dyes. the colors typically seen are pink for eosin, and dark red/purple/blue for hematoxylin. Generally used across histology and pathology. |
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| BASIC; stains anything that is acidic--e.g. nucleic acids of the nuclei and ribosomes. Gives a purple/dark red/blue color. |
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| an ACIDIC dye; labels lysine and arginine-rich proteins (which are basic amino acids) |
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| 3 dyes used together; these will label fibers of extracellular matrix rich in lysine and arginine as well as carbohydrates. Can also label cells enriched in protein and nucleic acids. Will give three distinct colors to specific substances. |
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| Purple substance when Masson's trichrome is used |
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| Blue/Green when treated with Masson's trichrome |
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| collagen or ground substance |
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| Red when treated with Mason's trichrome |
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| Periodic Acid-Schiff (PAS) |
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| stains carbohydrates; is very specific and will pretty clearly define glycocalyx and substances with mucus like the goblet cell of the small intestine |
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| will label fats in non-cleared tissues, such as those that were frozen. Can be used on adipocytes and myelin sheeth of neurons. |
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| Used to detect classes of enzymes like phosphatases, dehydrogenases, betacalactosidases, and ATPases. These techniques employ modified substrates that are converted to colored products via enzymes. Require frozen tissue that hasn't been cleared. |
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| antibody labeling; can be used to localize specific proteins or carbohydrates within a tissue. Antibodies bind specifically to the proteins or carbohydrates they are made agains; will link with detection molecules like a peroxidase or gold. This is frequently used in diagnostic pathology and can be used with paraffin tissues fixed by formalin; tissue is prepared for electron microscopy. |
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