Topic 1 Microscopy

Question 1

1. Stereomicroscope (light)
2. Compound Microscope (light)
3. Phase Contrast Microscope
4. Confocal Laser Scanning Microscope and Fluorescence
5. Scanning Electron Microscope (SEM)
6. Cryo SEM
7. Transmission Electron Microscope (TEM)
8. Electron Tomography

Answer 1

Microscopy Techniques

Question 2

• Uses visible light to view surface of sample
• Can view living samples
• Has low light resolution

Answer 2

Stereomicroscope (light)

Question 3

• Uses visible light to view thin section of sample
• Can view some living samples
• May require staining

Answer 3

Compound Microscope (light)

Question 4

• Uses light phases and contrast
• Can see internal structures if thin
• Good resolution and contrast
• Not good for thick samples, may produce “halo effect”

Answer 4

Phase Contrast Microscope

Question 5

• Used to observe thin slices
• Common for viewing chromosomes during mitosis
• Uses fluorescent tagging
• Causes artifacts
• Can be used without fluorescence

Answer 5

Confocal Laser Scanning Microscope and Fluorescence

Question 6

• View surface of 3D objects
• Cannot use on living samples
• Must be dried and coated

Answer 6

Scanning Electron Microscope (SEM)

Question 7

• Similar to SEM
• Sample not dehydrated
• Cannot use on living samples
• Must be frozen
• Can cause artifacts

Answer 7

Cryo SEM

Question 8

• Observe very thin cross-sections
• Can observe internal structures
• Cannot be used on living samples

Answer 8

Transmission Electron Microscope (TEM)

Question 9

• Not a type of microscope

- Technique used to build 3D model of sample using TEM data

Answer 9

Electron Tomography

Question 10

• Used to prepare a sample for observation or experimentation
• Spins and separates liquified cell homogenates into layers based on density
• Most dense separating first and least dense last
• In cells the order is: nuclei layer -> mitochondria/chloroplasts/lysosomes -> microsomes/small vesicles -> ribosomes/viruses/larger macromolecules
• Insoluble proteins can be found in the pellet
• Soluble proteins can be found in the supernatant, liquid above the pellet

Answer 10

Centrifugation

Question 11

1. Differential centrifugation:

2. Density centrifugation:

Answer 11

Types of Centrifugation

Question 12

• Spin pattern described earlier (spin, separate dense pellet, repeat)

Answer 12

Differential centrifugation:

Question 13

• Relies on density, shape, and speed at which macromolecule travels
• Forms continuous layers of sediment

Answer 13

Density centrifugation: