Electron Microscopy

Question 1

What size is the tissue cut on an ultramicrotome?

Answer 1

1 millimeter, then reduced to 0.5 millimeters to cut a section thickness from 50-100 nanometers.

Question 2

What are the 3 types of resins used in EM infiltration?

Answer 2

Epoxy, polyester, and methacrylates

Question 3

What media is used for EM embedding?

Answer 3

Resins. Commonly called plastics.

Question 4

Why are accelerators added to EM embedding resins?

Answer 4

To aid in polymerization.

Question 5

How is propylene oxide used in electron microscopy?

Answer 5

It is a transitional fluid. Transitional fluids take the place of clearing solutions in processing for plastic embedding. They make sure all the water is removed.

Question 6

True or False: Fixation for electron microscopy can be delayed longer than it can for light microscopy.

Answer 6

False. Fixation needs to be prompt and complete. Tissue degradation begins at the cellular level and affects visualizing ultrastructure (intercellular material) MORE than with morphologic changes at the tissue level.

Question 7

Electron microscopy is used to observe the ultrastructure of cells. What is the disadvantage of using aldehyde fixatives to look at cellular membranes?

Answer 7

Cell mebranes are composed of phospholipids. Aldehyde fixatives preserve lipids, but do not make them insoluble. Calcium Formalin is recommended for the preservation of phospholipids-apparently the Calcium Chloride helps in this regard.

Question 8

True or False: Osmium tetroxide stains as well as fixes tissue.

Answer 8

True. Fixing EM specimens with Osmium tetroxide gives great preservation of the plasmalemma and cytoplasm, but bothEM and LM (light microscopy) specimens can be stained with osmium tetroxide.

Question 9

Which EM buffer contains arsenic?

Answer 9

Cacodylate

Question 10

True or False: isotonic pH of fixatives is critical in EM.

Answer 10

True.

Question 11

Name 2 buffers used in EM fixation.

Answer 11

Phosphate, cacodylate, S-collidine, veronal acetate.

Question 12

What is the buffer used in Milloning fixative?

Answer 12

Phosphate

Question 13

What is the best buffer for Osmium Tetroxide?

Answer 13

S-Collidine

Question 14

Which buffer cannot be used with aldehyde fixatives?

Answer 14

Veronal Acetate

Question 15

True or False: It is easy to overdehydrate tissue when processing for EM.

Answer 15

False. Unlike FFPE tissue, when processing for EM complete dehydration, including bound water is necessary. This is due to the plastic embedding medium. If the resin doesn’t penetrate completely and polymerize, the specimen can melt under the electron beam.

Question 16

Would reagent alcohol be a good dehydrant choice for EM?

Answer 16

No. Reagent alcohol has been denatured with methanol and isopropanol, which will distort tissue structures. If alcohol is used as a dehydrant for EM, it must be pure ethanol.

Question 17

Name 2 dehydrants used in EM tissue processing.

Answer 17

Ethanol and Acetone.

Question 18

What step in EM processing comes between the dehydrating step and the infiltrating step?

Answer 18

Transitional fluid step. In FFPE processing this would be the clearing step. Transitional fluids continue dehydration and are miscible with the EM embedding medium (usually plastic).

Question 19

Name 2 transitional fluids.

Answer 19

Propylene Oxide and Styrene.

Question 20

How is correct orientation of ultrathin sections determined?

Answer 20

By cutting a semi-thin or thick section and staining it with toluidine blue or methylene blue.

Question 21

How can you tell if an ultrathin
EM section is the right thickness

Answer 21

The color reflected from both the waterbath and the section gives an interference color which will tell you the thickness of the section.

Question 22

True or False: EM sections are mounted on copper grids and stained with heavy metals to obtain a micrograph.

Answer 22

True. The metals are electron dense and increase the contrast of the projection.

Question 23

Name 2 EM stains.

Answer 23

Lead, Urynal, Silver, Phosphotunstic acid, Potassium Permanganate, Bismuth, Thorium, Indium, Vandium, Lanthanum

Question 24

True or False: A light microscope bends light beams with glass lenses and an electron microscopes bend an electron beam through electromagnetic lenses.

Answer 24

True. An electron gun generates electrons. In a vacuum, accelerated electrons travel in wavelengths like light, though shorter, and can be directed by electromagnetic lenses.