Microscopy

Question 1

How do you get the final magnification of a microscope?

Answer 1

multiply the magnification of the objective lens by the magnification of the projection lens

Question 2

What is the resolution?

Answer 2

the ability to distinguish between two objects

Question 3

How is resolution calculated?

Answer 3

D= 0.61 (wavelength)/Nsina

smaller number=higher resolution

Question 4

What are the steps of sample preparation?

Answer 4

1. fixation
2. embedding
3. sectioning
4. staining

Question 5

What does the fixation step of sample preparation do?

Answer 5

• cross-links macromolecules and partially permeabilizes cells for staining
• uses cross-linking agents that form covalent bonds with free amino groups: glutaraldehyde and formaldehyde

Question 6

What does the embedding step of sample preparation do?

Answer 6

• places sample in wax or resin

- only for thick specimens

Question 7

What does the sectioning step of sample preparation do?

Answer 7

• cuts embedded sample into thinner sections so light can pass through
• uses a sectioning apparatus called a microtome

Question 8

What does the staining step of sample preparation do?

Answer 8

• gives the sample color to give it contrast

- ex: hematoxin, eosin, Wright Glesma, benzidine

Question 9

What are the advantages of Brightfield microscopy?

Answer 9

• simple
• inexpensive
• no staining required
• live cell imaging in cell culture (1-2 cell layer samples)

Question 10

What are the limitations of Brightfield microscopy?

Answer 10

• very low contrast

- low apparent optical resolution

Question 11

What do you need to add to a Brightfield microscope to make it a phase contrast microscope?

Answer 11

• phase plate

- annular diaphragm