Electron Mcroscopy Flashcards
What is an Ultramicrotome?
A special microtome that produces ultrathin, electron transparent slices of tissue roughly 90nm thick for electron microscopy
Sits on an anti-vibration bench to prevent poor sections
Automatic, super precise arm movements of 70-130nm, and super expensive as a result, also has microscope oculars so you can see what you are doing
Tiny water boat/trough built in to catch the sections
EM Dehydration process
Removes water and other potentially volatile components (via displacement with an organic solvent) that would vaporize under the immense heat conditions caused by the electron beam
Fixative is rinsed out/replaced with an equal pH buffer, buffer is removed with distilled water to remove residual salts that could effect the resin, then water is removed with graded alcohol or acetone, then replaced with organic solvent
Tends to cause cell shrinkage but is necessary
Allows for infiltration and embedding with appropriate resinous media
EM infiltration process
Sample is essentially perfused with pure resin polymer (not miscible with water, so dehydration is essential) that replaces the organic solvent (can take several hours), can’t have bubbles or water left behind, to make the sample stable enough for electron bombardment
epoxy resins are the most popular because they cross-link with themselves and the fixed tissue
Also the most toxic and carcinogenic
Ultrathin sectioning on the ultramicrotome
Ultrathin sections cut from the polymerized resin block using a glass blade to face in and a $3k diamond blade for the final individual section
Glass knife/blade
For facing in, ground by hand, cheap
good for a week if stored in a dust free environment
Diamond knife/blade
For taking the final sections, obtained via company exchange system, $3k per blade
Lubricated with water from the water bath/boat while cutting to reduce pressure on the specimen
Embedment
Sample must be completely infiltrated by pure resin (like gooey honey), then placed in the appropriate mold (usually bullet shaped) and polymerized in a special oven for approx 8 hours. (you can’t hold the sample in place while it freezes like with paraffin, the mold holds the sample in the proper orientation)
EM Polymerization process
Critical for resin to achieve proper hardness (chemical cross-linking) and tissue support; incorrect polymerization makes sectioning impossible
A specific hardener is in each resin kit unique to that particular resin’s composition and critical to proper hardening and polymerization
Which fixatives are used for EM
Glutaraldehyde primary followed by Osmium Tetroxide secondary
What does glutaraldehyde do?
Fixes proteins and carbohydrates in the tissue similarly to formaldehyde, just with an extra aldehyde, good penetration
Additive, non-coagulant fixative
What does osmium tetroxide do?
The osmium atoms serve as an electron stain giving contrast to the specimen, and are best at fixing lipids while being bad at fixing proteins and carbs
Poor penetration requiring small sample
What reagents are used to stain EM specimens?
Stains with low atomic weight that are not significantly different from the resins they are embedded in are used for the tissue
Then heavy metals are used as contrast against the resin and to selectively make certain tissue elements electron “opaque”
Criteria to examine a sample with EM
Complete lack of water or other volatile componants
Resistant to high vacuum conditions (no warping, etc)
Thermal and physical stability when exposed to the electron beam
Regions of electron opacity and transparity
Appropriate size for TEM (very tiny specimen)
Criteria to examine a sample with EM
Complete lack of water or other volatile components
Resistant to high vacuum conditions (no warping, etc)
Thermal and physical stability when exposed to the electron beam
Regions of electron opacity and transparity
Appropriate size for TEM (very tiny specimen)
What types of embedding media are used in EM?
Resins; essentially special super hard plastic to support the specimen for electron bombardment
