Unit 2: Cells

Question 1

Why do light microscopes have a lower resolution than electron microscopes?

Answer 1

Light rays have a longer wavelength than electrons.

Question 2

Define the resolution of a microscope.

Answer 2

The minimum distance apart two objects can be in order for them to be viewed as separate items.

Question 3

Resolution of light microscopes?

Answer 3

0.2um

Question 4

Resolution of electron microscopes?

Answer 4

0.1nm

Question 5

What is cell fractionation?

Answer 5

The process where cells are broken up and the different organelles they contain are separated out

Question 6

What is the purpose of cell fractionation?

Answer 6

To study the structure and function of various organelles that make up cells.

Question 7

Before cell fractionation can begin what is the tissue placed in?

Answer 7

A:
•Cold
•Isotonic (same water potential as tissue)
•Buffered
Solution

Question 8

Explain why the solution before cell fractionation requires specific conditions.

Answer 8

Being cold reduces enzyme activity that might break down organelles. Being isotonic prevents organelles bursting or shrinking as a result of osmotic gain or loss of water. Being buffered is so that the pH doesn’t fluctuate as a change in pH could alter the structure of organelles or affect the functioning of enzymes.

Question 9

What are the two stages of cell fractionation?

Answer 9

Homogenation and ultracentrifugation.

Question 10

Describe the homogenation process

Answer 10

Cells are broken up by a homogeniser (blender). This releases organelles from the cell. The resultant fluid, known as homogenate is then filtered to remove any complete cells and large prices of debris.

Question 11

Describe the ultracentrifugation process.

Answer 11

The tube of filtrate is placed in a centrifuge and spun at a low speed. The heaviest organelles, the nuclei are forced to the bottom of the tube where they form a thin sediment or pellet. The fluid at the top of the tube (supernatant) is removed, leaving just the sediment of the nuclei. The supernatant is transferred to another tube and spun in the centrifuge at a faster speed than before. The next heaviest organelles, the mitochondria are forced to the bottom of the tube. The process is continued so that with each increase in speed, the next heaviest organelle is sedimented and separated out.

Question 12

Why do electrons being negatively charged give the electron microscopes an advantage?

Answer 12

The beam can be focused using electromagnets.

Question 13

Why does the electron microscope require vacuum conditions?

Answer 13

Because electrons can be absorbed or deflected by the molecules in air.

Question 14

How does the TEM work?

Answer 14

An electron gun produces a beam of electrons that is focused onto the specimen by a condenser electromagnet. Parts of the specimen absorb the electrons and appear dark, other parts allow the electrons to pass through so appear bright.

Question 15

Why can the 0.1nm resolving power of the TEM not always be achieved in practise?

Answer 15

•Difficulties preparing the specimen.
•A higher energy electron beam is required and this may destroy the specimen.

Question 16

What are the main limitations of the TEM?

Answer 16

•Vacuum required so living specimens cannot be viewed.
•A complex staining process is required and the image is not in colour.
•The specimen must be extremely thin.
•The image may contain artefacts (things that result from the way a specimen is prepared but are not part is the natural specimen).

Question 17

What kind of image does a TEM produce?

Answer 17

Flat, colourless, 2D image.

Question 18

How can the image be made 3D in a TEM?

Answer 18

By taking a series of sections through a specimen and looking at the series of photomicrographs produced.

Question 19

Which limitations of the TEM do not apply to the SEM?

Answer 19

Sections do not need to be thin.

Question 20

Which limitations of the TEM do not apply to the SEM?

Answer 20

Specimens do not need to be extremely thin, as electrons do not penetrate.

Question 21

How does a SEM work?

Answer 21

It directs a beam of electrons onto the surface of the specimen from above. The beam is then passed back and forth across a portion of the specimen in a regular pattern. The electrons are scattered by the specimen and the pattern of this scattering depends on the contours of the specimen surface. We can then build up a 3D image by computer analysis of the pattern of scattered electrons and secondary electrons produced.

Question 22

What is the basic resolution of the SEM?

Answer 22

20nm (lower than the TEM).