Lecture 1: Techniques in Cell Biology

Question 1

Limit of resolution

Answer 1

• The minimum distance two objects can approach one another and still appear separate

Question 2

Light Microscopy

Answer 2

• Uses light (obviously) to view samples
• Limit of resolution for a light microscope is about half the wavelength of light
• This is because the object has to interfere with the wavelength in order to be seen
• If the object is so small the the wavelength kind of just glosses right over it, it can’t be seen (see 4/2 notes for pictures)
• Since visible light has a wavelength between .4-.7 u, the limit of resolution is between .2-.35 u
• This is why the smallest things that can be observed with a light microscope are mitochondria and small bacteria

Question 3

Factors that are important if an object is to be seen

Answer 3

1. Magnification

2. Resolution

Question 4

Problems with Light Microscopy and their solutions

Answer 4

1. Cells are about 70% water, so they are transparent
   - Solution: use stains and dyes for different parts of the cell, like DNA, proteins, membranes, etc
   - The dyed material can reflect light
2. Cells are fragile and can become distorted or broken open by observation, such as when a coverslip is placed on them
   - Solution: Fix Cells before observing them
3. Tissues are thick, so light can’t penetrate them, meaning they can’t be seen, and just appear as dark blobs
   - Solution: Embed the tissue in something stronger, such as paraffin wax or resin, and then slice it with a microtome
   - Microtomes are basically like deli slicers, but for tissues, and are able to slice the tissue into slices ranging from 1-10 u thick
   - The result is a ribbon of sections, which can be placed on a glass slide, stained, and then mounted under a cover slip for observation.
4. Cells are killed by staining, fixing, or sectioning, which can lead to us observing an “artifact,” which is essentially just a dead cell
   - Solution: Want to confirm your results by looking at a cell you know is living, which utilizes interference microscopy (still light microscopy) and other technology that is still developing

Question 5

Fixation

Answer 5

• Used to help stabilize/preserve cells
  There are two different types of fixation
  1. Methanol fixation
• causes proteins to denature and precipitate our in place, complexed to one another
  2. Chemical Crosslinkers
• Can be either formaldehyde or glutaraldehyde
  -Have charged groups on their ends to they can bind to proteins, effectively linking them together by acting as the “bridge”
• Glutaraldehyde binds to the lysines of proteins

Question 6

Interference Microscopy

Answer 6

• A type of light microscopy used to look at living cells