Microscopes

Question 1

What is the formula for magnification?

Answer 1

Size of Image / Size of Real Object

Question 2

What is resolution?

Answer 2

Resolution/Resolving Power, is the minimum distance apart that two objects can be in order for them to appear as separate items.

Question 3

What is cell fractionation?

Answer 3

Where cells are broken up, and the different organelles they contain are separated out.

Question 4

Why is the solution kept cold?

Answer 4

To reduce enzyme activity that might break down the organelles.

Question 5

Why is the solution kept isotonic?

Answer 5

To keep the same water potential as the original tissue, it is to prevent organelles bursting or shrinking as a result of osmotic gain or loss of water.

Question 6

Why is the solution buffered?

Answer 6

So that the pH does not fluctuate, any changes in pH could alter the structure of the organelles, or effect the functioning of the enzymes.

Question 7

What are the two stages of cell fractionation?

Answer 7

• Homogenation

* Ultracentrifugation

Question 8

What is the process of homogenation?

Answer 8

• Cells are broken up by a homogeniser
• The organelles are released from the cell, and the resultant fluid containing it is called the homogenate.
• It is filtered to remove debris and complete cells.

Question 9

What is the process of ultracentrifugation?

Answer 9

• Tube of filtrate is placed in the centrifuge and spun at a slow speed
• Heaviest organelles, the nuclei are forced to the bottom of the tube, where they form a thin sediment.
• The fluid at the top of the tube, the supernatant, is removed, leaving just the sediment of nuclei.
• Supernatant transferred to another tube and spun in the centrifuge at a faster speed than before.
• Next heaviest organelles, are forced to the bottom of the tube,.
• Process repeated until desired organelle forms a pellet at the bottom.

Question 10

Why were electron microscopes used over light microscopes?

Answer 10

• Electron beams have a very short wavelength, and the microscopes can therefore resolve objects well, high resolving power.
• Electrons are negatively charged, so the beam can be focused using electromagnets.

Question 11

What are the two types of electron microscope?

Answer 11

• Transmission Electron Microscope

* Scanning Electron Microscope

Question 12

How do Transmission electron microscopes work?

Answer 12

• Electromagnets are used to focus a beam of electrons which is transmitted through the specimen.
• Denser parts of the specimen absorb more electrons, which makes them darker on the image you end up with.

Question 13

Why can the true resolving power not be achieved in practice?

Answer 13

• Difficulties preparing the specimen limit the resolution that can be achieved.
• A higher energy electron beam is required, this may destroy the specimen.

Question 14

What are the limitations of TEMs?

Answer 14

• A vacuum is required, therefore living specimens cannot be observed.
• A complex staining process is required, and the image is not in colour.
• Specimen must be extremely thin.
• The image may contain artefacts

Question 15

What are artefacts?

Answer 15

• Things that result from the way the the specimen is prepared, artefacts are not a part of the specimen and may appear on the finished image.
• It results in difficulties telling whether a specific item is an artefact or a part of the specimen.

Question 16

What are some advantages of TEMs?

Answer 16

High resolution images, so you can see the internal structures of thin organisms.

Question 17

How do SEMs work?

Answer 17

• A beam of electrons is scanned across the specimen which knocks off electrons from the specimen, which are gathered in a cathode ray tube to form an image.

Question 18

What are some advantages of SEMs?

Answer 18

• Images show the surface of the specimen, and can be 3-D.

* Can be used on thick specimens.

Question 19

What are some of the drawbacks of SEMs?

Answer 19

• A vacuum is required, therefore living specimens cannot be observed.
• A complex staining process is required, and the image is not in colour.
• The image may contain artefacts
• A vacuum is required, therefore living specimens cannot be observed.
• Lower resolving power than TEMs

Question 20

How do light microscopes work?

Answer 20

• They use light to form an image.

Question 21

What is a disadvantage of light microscopes?

Answer 21

• Low resolving power, cannot view small organelle internal structures.
• Low maximum useful magnification.