Terms in this set (20)
What are the features of the Electron Microscope? How do we prepare the samples according to those features?
- Higher resolving power - Better fixation
- Low penetration of the beam - Thin section needed
- High vacuum - dehydrated, non-living samples needed
- Scattering image - Heavy metal staining needed
Describe the steps to prepare for electron microscopy
- material collecting
- Use of transitional solvent
Describe the fixation of the Electron Microscope
- Double fixation is used
- Glutaraldehyde is first used (protein-cross linker)
- Osmium tetroxide is then used (preserve and stain the membrane; increase the contrast)
Describe how we put fixatives on the samples
- dripping a drop of fixative on the organ
- immersion of the dissected tissue into the solvent
How is the thin sectioning done in Electron Microscopy?
- Use of ultramicrotome with the diamond knife
- Thickness of the sample is between 50-100nm
What is used in the post-staining?
- uranyl (nucleic acids) and lead salts (proteins) are used to increase the contrast achieved by osminum during the fixation
Describe semi-thin sectioning
- use of plastic box instead of paraffin so that thinner sections can be cut
- provide the greater definition of the cellular details than the paraffin-boxed sample
- Only small size of sample can be viewed
Compare thick, semi-thin, and thin sectioning.
When do we use suspension techniques?
- When the sample is not too thick, it can be applied without sectioning. This is the situation during study isolated organelles or bacteria, supra-molecular complexes (eg. ribosomes, chromatin, viruses) and macromolecules (polysaccharides, proteins). There are two types of suspension techniques - negative staining and heavy metal shadowing
What is negative staining?
- a quick method for checking the quality of fraction during the isolation of particular cell components.
- The principle is the application of heavy metal salt solution, which dries down around the specimen on a support film coated grid.
- the specimen itself remains electron-translucent whilst its immediate surrounding does not
What is metal shadowing?
- the contrast is achieved by depositing atoms of heavy metals (platinum, tungsten) by vacuum evaporation at an acute angle for depicting isolated macromolecules (e.g., nucleic acids, chromatin)
What are the steps of production and the clearing of the carbon-metal replica that is used in freeze-fracturing?
What is scattering image?
- It refers to the scattered electrons that are filtered by the aperture of the objective lens and do not contribute to the image formation
Why does the Electron Microscopy have better resolution than the Light Microscopy?
- The wavelength of an electron beam is smaller than that of light; so replacing light by electron beam gives better resolution.
What is Scanning Electron Microscope?
- It is a type of an electron microscope that permits the pseudo-3-Dimensional views of the surface of the unsectioned cells, tissues, organs, and small animals.
How does scanning electron microscope form the image?
the secondarily emitted or backscattered electrons are detected, and the image of specimen appears on a CTR (cathode ray tube) screen.
How does the structure that scatters the electrons appear?
- Dark. Appears white if the object absorbs the electron
How do we increase the resolution of the EM?
- By increasing the voltage of the anode.
What does uranyl stain?
- nucleic acid
What does lead salt stain?
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