Electron Microscopy Flashcards
The electron microscope has a higher magnification range than the light microscope.
General Microscopy:
- Upper limit of useful magnification with the light microscope is about 1,000 diameters (x).
- Electron microscopy has a magnification range from approximately 1,000 to 500,000 diameters.
How does the electron microscope obtain extra resolving power?
What emits the electrons?
What is the source?
The electron gun, electron beam, and specimen are all kept under what?
◦ The electron microscope obtains extra resolving power by replacing the ordinary light source of a light microscope with an “electron gun”.
Electrified tungsten filament that emits
electrons.
The electron gun (source), electron beam
& specimen are all kept under vacuum.
What are the two major types of electron microscopes
1.) Transmission electron microscope (TEM)
2.) Scanning electron microscope (SEM).
Describe the specimen under a Transmission Electron Microscope.
What type of image does this method produce?
Specimen (usually a very thin section) that
will either:
1.) Transmit electrons – producing a “clear” area of the image
2.) Deflect electrons – producing a “dark” area of the image.
This method produces a two-dimensional
black and white image.
Describe how the Scanning Electron Microscopy operates.
what type of image does this method produce?
What is the difference between a TEM & SEM?
Has an electron beam that “sweeps” the surface of the specimen.
This method produces a three-dimensional image of the specimen.
◦ SEM does not have the high magnification of TEM, but can provide images of extreme detail and focus.
Describe Negative Staining.
Describe Positive Staining.
Negative Staining: interaction between particles
show up bright on a dark background.
- Examples: mainly for bacteria, and cell
fragments
Positive Staining: interaction between particles
show up dark on a bright background
- Examples: Uranyl Acetate
Fixation is crucial for tissue designated for what?
Why is fixation crucial in cytology/EM? (2 things)
What will good fixation demonstrate? (5 things)
Crucial for tissue designate for EM.
1.) Must preserve the “ultra-structures” of tissue.
2.) Cellular level fixation is very apparent
◦ Good fixation will demonstrate:
1.) Complete plasmalemma (no break in membrane)
2.) Nuclear envelope will appear uniform
3.) Mitochondria (no swelling or disruption)
4.) Endoplasmic Reticulum
5.) Cytoplasm & Nucleus
What are the two fixation categories for general use?
1.) Osmium Tetroxide provides superior cytological detail
2.) Aldehydes for their general fixative properties
What are the three major fixatives used in Electron Microscopy?
1.) Osmium Tetroxide fixative
2.) Aldehyde fixation
- Formaldehyde
- Glutaraldehyde
- Paraformaldehyde (pure formaldehyde)
3.) Zamboni Picric acid – Formaldehyde (PAF) fixative
Advantages of Osmium Tetroxide fixation? (2 things)
Disadvantages of Osmium Tetroxide Fixation? (3 things)
Advantages:
1.) Provides excellent cytological preservation
2.) Renders lipids insoluble, giving excellent membrane preservation.
Disadvantages:
1.) Cannot exceed 4 hours in fixative
2.) Penetration poor
3.) NO IHC can be done post Osmium Tetroxide fixation
Advantages of Primary Aldehyde Fixation? (4 things)
Disadvantages of Primary Aldehyde Fixation (2 things)
Advantages:
1.) Allows better penetration
2.) When followed by postosmication (secondary fixation in osmium tetroxide) optimum preservation.
3.) Can be dual-purpose (light and EM)
4.) IHC can be done with these fixatives
Disadvantages:
1.) Lipids not preserved (without post osmium)
2.) Possible swelling of membrane-bound cavities.
Advantages of Zamboni PAF Solution? (3 things)
Disadvantages of Zamboni PAF Solution (2 things)
Advantages:
1.) Specimens can remain in fixative indefinitely
2.) Penetrates tissue rapidly and stabilizes cellular proteins.
3.) Can be used for both light and EM.
Disadvantages:
1.) Lipids are not well preserved (without secondary osmium)
2.) Some cytoplasmic granules and lysosomes may not be preserved.
Factors influencing fixation:
(They are similar, if not the same as standard histological fixation)
1.) pH: for electron microscopy, fixatives are
usually buffered between 7.2-7.4. Buffers used
are phosphate, cacodylate, s-collidine, and
veronal acetate
2.) Temperature: traditionally done at 4C, this however destroyed perinuclear membrane and microtubes. It has since been done at room temperature
Factors influencing fixation
3.) Tonicity: blood plasma has a tonicity of around 300 mOsm, which many believe to be the best osmolality. Sucrose and dextrose are used to adjust the tonicity
4.) Length: some fixatives have strict time frames for fixation. Most aldehyde fixatives can hold tissues indefinitely. Glutaraldehyde however should only be used overnight at most, 2-4 hours is recommended
What is significant about Dual purpose fixatives?
Name some Dual purpose fixatives.
They eliminate the need to preselect
specimens prior to fixation for electron
microscopy, as they perform well for both
electron microscopy, and routine histology
Include: modified Millonig formaldehyde,
formaldehyde AND glutaraldehyde, along with
buffered PAF solution (Zamboni)
