FINALS - 2 - TEM

Question 1

What is the definition of Electron Microscopy?

Answer 1

Uses a beam of electrons (instead of light) to create an image

Question 2

What is the source of electrons in Electron Microscopy?

Answer 2

Tungsten filament (heated to emit electron beam)

Question 3

What is the primary purpose of Electron Microscopy?

Answer 3

Used for ultrastructural study; provides higher resolution and magnification

Question 4

What is the maximum magnification of Transmission Electron Microscopy (TEM)?

Answer 4

Up to 2,000,000x

Question 5

What is the resolution of Transmission Electron Microscopy (TEM)?

Answer 5

0.2 nanometers

Question 6

What types of lenses are used in Electron Microscopy?

Answer 6

• Electromagnetic * Electrostatic

Question 7

How is the electromagnetic lens designed?

Answer 7

Solenoid – coil through which electron beam passes

Question 8

What are the main parts of a Transmission Electron Microscope (TEM)?

Answer 8

• Electron source (tungsten) * Electromagnetic lens system * Sample holder * Imaging system (screen with phosphorescent plate)

Question 9

When is Transmission Electron Microscopy (TEM) used diagnostically?

Answer 9

When structural/functional abnormalities aren’t visible on light microscopy

Question 10

Give an example of a disease that requires TEM.

Answer 10

Minimal change disease in glomerulonephritis – looks normal on LM

Question 11

When should TEM be used in testing ambiguity?

Answer 11

When affinity labeling results are equivocal

Question 12

When is TEM used when no alternatives exist?

Answer 12

When no simpler test is available

Question 13

What are other uses of TEM?

Answer 13

Research on new diseases or microorganisms

Question 14

What are the full processing steps for TEM?

Answer 14

• Primary fixation (aldehyde) * Rinsing * Secondary fixation (osmium) * Dehydration * Resin embedding * Trimming * Ultramicrotomy * Staining * Grid mounting * Imaging

Question 15

What is the primary fixative used in TEM?

Answer 15

2.5% glutaraldehyde in 100 mM phosphate buffer, pH 7.0

Question 16

What is the fixation temperature sequence for TEM?

Answer 16

Room temp for 15–30 mins, then 4°C for stability

Question 17

What is the maximum tissue size for fixation in TEM?

Answer 17

1 mm³

Question 18

What is the purpose of fixation in TEM?

Answer 18

Cross-links cellular structures to preserve morphology and spatial relationship

Question 19

What is the purpose of rinsing after fixation in TEM?

Answer 19

Removes aldehyde to prevent precipitate with osmium

Question 20

What is the recommended buffer for rinsing in TEM?

Answer 20

Sodium cacodylate (pH 5.1–7.4)

Question 21

What buffer should be avoided with aldehydes during rinsing?

Answer 21

Veronal buffer (contains barbitals)

Question 22

What is the post-fixative used in TEM?

Answer 22

1% osmium tetroxide in 100 mM phosphate buffer

Question 23

What is the temperature and time for post-fixation in TEM?

Answer 23

1–2 hours at 4°C

Question 24

What is the purpose of post-fixation in TEM?

Answer 24

Increases stability and contrast of fine structures

Question 25

What is the preferred dehydration agent in TEM?

Answer 25

Acetone (less lipid loss than ethanol)

Question 26

What is the dehydration sequence in TEM?

Answer 26

* 30% * 50% * 70% * 90% * 100% acetone, gradually increased duration

Question 27

What are the resins used for embedding in TEM?

Answer 27

* Epoxy * Polyester * Methacrylate

Question 28

What is the purpose of resin infiltration in TEM?

Answer 28

Creates hard matrix to allow thin sectioning

Question 29

What is the polymerization process for resin in TEM?

Answer 29

Room temp overnight + oven at 60°C for 2–3 days

Question 30

How can the quality of polymerization be tested in TEM?

Answer 30

Press capsule ridge with fingernail to check hardness

Question 31

What is the purpose of trimming the capsule in TEM?

Answer 31

Expose pyramid tip of tissue

Question 32

What tools are used for trimming in TEM?

Answer 32

Old glass knives

Question 33

What is the ideal angle of pyramid facets in TEM trimming?

Answer 33

About 45°

Question 34

What is the typical section thickness in TEM?

Answer 34

30–60 nm

Question 35

How are sections collected in TEM?

Answer 35

Float on water surface, collected on copper grids

Question 36

What stain is used for general observation in TEM?

Answer 36

Toluidine blue

Question 37

What is the purpose of positive staining in TEM?

Answer 37

Adds electron-dense material to highlight structures (dark on light background)

Question 38

Give examples of positive stains used in TEM.

Answer 38

* Uranyl acetate * Lead citrate

Question 39

What is the purpose of negative staining in TEM?

Answer 39

Stains background so objects appear light (light on dark)

Question 40

Give examples of negative stains used in TEM.

Answer 40

* Uranyl acetate * Uranyl formate * Ammonium molybdate * Phosphotungstic acid

Question 41

What is the function of Lead citrate in TEM?

Answer 41

Best general stain, works at high pH, stains nuclei, membranes, glycogen, etc.

Question 42

How can Uranyl acetate be used in TEM?

Answer 42

Can be used during dehydration or as aqueous stain

Question 43

What is the use of Phosphotungstic acid in TEM?

Answer 43

Used as block stain during dehydration (1–2% aqueous for 30 mins)