Lecture 3- Methods in Cell BIo

Question 1

Are there cells that can be propagated forever?

Answer 1

1. Stem cells
2. Cancer cells

Question 2

What are techniques used in cell bio?

Answer 2

1. Light microscopy
2. Fluorescence microscopy
3. Laser Scanning Confocal Microscopy
4. Scanning Electron Microscopy (SEM)
5. SDS-Polyacrylamide Gel Electrophoreses (PAGE)
6. Western Blotting
7. X-ray crystallography and NMR spectroscopy

Question 3

Which of these cells will be more appropriate to study the nuclear components in cells?

a. Red blood cells
b. Liver cells

Answer 3

a. Red blood cells
- Red blood cells don’t have nuclei

Question 4

Light microscopy

Answer 4

• Employs visible light to detect small objects

Question 5

Explain the difference between maginification and resolution.

Answer 5

Magnification- ability to make small objects seem larger, such as making a microscopic org. visible

Resolution- ability to distinguish 2 objects that are close from each other

Question 6

How can you improve the ability to see details in cells using light microscopy?

Answer 6

a. Phase contrast
b. Digital-interference- contrast microscopy

• Takes advantage o/ the diffs. in refractive index and thickness o/ cellular material
• Difference in image you see depends on how you light the samples; usually through filters and polarized light
• Produces images that reveal diff. features o/ cell architecture

Question 7

Explain Phase contrast and Digital-interference- contrast microscopy.

Answer 7

Phase contrast- good for location and movement o/ larger organelles in live cells in thin layers
- Convert phase shifts in light passing through a transparent specimen to brightness changes in the image

DIC- visualizing extremely small details and thick objects

Question 8

You’re still not happy with the details you’re able to see using bright field microscopy. How can you highlight different regions of the cell better?

Answer 8

Use stains in tandem w/ bright field microscopy
1. Fixation- cross link proteins using chemicals to preserve cellular morphology
2. Obtain a thin section
3. Permeabilize cells to dyes (uses detergent)
4. Stain

Question 9

How are hematoxylin and eosin (H&E) stains used in bright field microscopy?

Answer 9

Hematoxylin- positively charged - binds DNA - BLUE COLOR
Eosin- negatively charged - binds basic amino acids - stains cytoplasm and extracellular matrix - PINK COLOR

Question 10

Fluorescence microscopy

Answer 10

• Specialized type o/ light microscopy
• Phenomena where materials absorb light o/ one (shorter) wavelength, then re-emit light o/ a diff. (longer) wavelength
• Light source = White light
• Excitation filter: Allows only desired wavelength to pass and illuminate the sample
• Dichroic mirror- reflect excitation light into sample
• Allows longer wavelength emitted light to pass
• Emission filter- used to detect only 1 wavelength o/ light coming back from specimen

Question 11

T/F: Fluorescence microscopy has the same limit of resolution as white light microscopy!

Answer 11

True

Question 12

What are the pros of fluorescence microscopy?

Answer 12

1. Can detect intracellular Ca2+ levels in cells
2. Can detect specific proteins in fixed cells

Question 13

Fluorescent Resonance Energy Transfer (FRET)

Answer 13

Pros
- Good for determining how close together two molecules are in a cell (has to be closer than 10 nm)
- To follow the activation o/ an enzyme “in situ”

Cons
- Cell can look “fuzzy” (cell has layers)

Question 14

You took fluorescent images o/ a cell but it looks a bit fuzzy. How can you overcome this?

Answer 14

Laser Scanning Confocal Microscopy

• Similar to fluorescent microscopy, but uses lasers to light one color
• You look at one focal plane at a time by taking optical sections through the cell
• Eliminates out o/ focus light