Cell Structure - Methods Of Studying Cells

Question 1

What is the resolving power of light microscopes and why is this?

Answer 1

0.2 micrometers (quite a large distance between two points) because of the relatively long wavelength of light rays

Question 2

What is the resolving power of electron microscopes and why is this?

Answer 2

0.1 nm (short distance between two points) because electron microscopes use electron beams which have a lot shorter wavelengths than visible light rays.

Question 3

What is the formula for magnification?

Answer 3

Magnification = image size / actual size (being the size of the object)

Question 4

What is the definition of magnification?

Answer 4

How much bigger the image is compared to the actual size of the object

Question 5

What is the definition of resolution?

Answer 5

The smaller distance points can be separated and still be seen as separate items. (The amount of detail an image appears in depends on the microscopes resolving power)

Question 6

What limits resolution?

Answer 6

The wavelength of light used by the microscope

Question 7

What does the electron beams wavelength mean for the resolving power of the electron microscope?

Answer 7

The electron beam has a very short wavelength so resolves objects well so has a high resolving power

Question 8

Why is a near-vacuum created within the chamber of an electron microscope for it to work? What does this mean for objects that are being examined under an electron microscope?

Answer 8

Because electrons would otherwise be absorbed or deflected by molecules in the air and would never reach the specimen. This means that objects being seen under an electron microscope have to be dead

Question 9

Describe the radiation pathway in an electron microscope

Answer 9

Starting from the electron source, the electron beam,

1. Goes through a magnetic condenser which goes through the object
2. It then goes through the magnetic objective to produce the immediate image
3. The electron beam then goes through a magnetic projector which projects the image on a fluorescent screen so the object can be observed

Question 10

What are the two types of electron microscope

Answer 10

• The transmission electron microscope

- the scanning electron microscope

Question 11

What is a photomicrograph?

Answer 11

An image produced by a transmission electron microscope which has been photographed from a screen

Question 12

How does a transmission electron microscope work?

Answer 12

1. An electron gun produces a beam of electrons which is focussed onto the specimen by a condenser electromagnet
2. The beam then passes through a thin section of the specimen where parts of the specimen absorb electrons and appear dark while other parts allow electrons to pass through and appear bright
3. An image is then produced on a screen which can be photographed to give a photomicrograph

Question 13

The resolving power of a transmission electron microscope is 0.1nm but can’t always be achieved in practice. Why?

Answer 13

• difficulties in preparing the specimen limit the resolution that can be achieved
• a higher energy electron beam is required and this may destroy the specimen

Question 14

What are the main limitations of an electron microscope?

Answer 14

• The whole system MUST be in a vacuum and so living specimens can’t be observed
• a complex staining process with heavy metals is required and even then the image isn’t in colour
• the specimen must be extremely thin
• the image may contain artefacts so it’s not always easy to be sure that what is seen on a photomicrograph really exists in that form

Question 15

What are artefacts?

Answer 15

In transmission electron microscopes, they are the result of the way the specimen is prepared that may appear on the finished photomicrograph that are not part of the natural specimen.

Question 16

Why is a flat 2D image produced in a transmission electron microscope?

Answer 16

Because the specimen must be extremely thin in order for electrons to penetrate

Question 17

How is a the way a scanning electron microscope works different to a transmission electron microscope?

Answer 17

1. The scanning electron microscope directs a beam of electrons from electrons onto the surface of the specimen from above rather than penetrating the specimen in the transmission microscope
2. The beam is then passed back and forth across a portion of the specimen in a regular pattern
3. The electrons are then scattered by the specimen and the the pattern of scattering depends on the contours of the specimens surface
4. A 3D image can then be built up by computer analysis of the pattern of scattered electrons and secondary electrons produced.

Question 18

Compare the resolving power of a transmission electron microscope and a scanning electron microscope

Answer 18

The transmission electron microscope has a resolving power of 0.1nm and the scanning electron microscope has a lower resolving power of 20nm

Question 19

Compare the thickness of the specimen between the transmission electron microscope and the scanning electron microscope

Answer 19

The transmission electron microscope needs the specimen to be very thin as electrons have to penetrate where as the scanning electron microscope doesn’t need the specimen to be very thin as the electrons do not penetrate but instead are passed on the surface

Question 20

What is an eyepiece graticule and what does it do?

Answer 20

In a light microscope, it’s a glass disk that has a scale typically 10mm long and divided into 100 subdivisions that’s placed in the eyepiece of a microscope and can allow you to measure the size of objects

Question 21

Where can you get more information on calibrating the eyepiece graticule and measuring cells?

Answer 21

YouTube :)

Question 22

What is the endoplasmic rectilium

Answer 22

A large 3D system of sheet like membranes spread through the cytoplasm of cells and is continuous with the outer nuclear membrane

Question 23

What are cisternae

Answer 23

A network of tubules and flattened sacks enclosed by the membrane of the ER

Question 24

What is cell fractionation?

Answer 24

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

Question 25

Before cell fractionation begins why is the solution

• cold
• the same water potential as the tissue
• buffered?

Answer 25

• to reduce enzyme activity that might break down organelles
• to prevent organelles bursting or shrinking as a result of osmotic gain or loss of water
• to prevent the pH fluctuating as any change in pH could alter the structure of the organelles or affect the functioning of the enzymes

Question 26

What are the two stages of cell fractionation?

Answer 26

Homogenation and ultracentrifugation

Question 27

In cell fractionation, what is homogenation? Why is it carried out?

Answer 27

where cells are broken up by a homogeniser (blender). And the homogenate (the resultant fluid) is filtered to remove any complete cells and large pieces of debris. This is to release the organelles from their cells

Question 28

In cell fractionation, what is ultracentrifugation?

Answer 28

The process by which the fragments in the filtered homogenate are separated in a centrifuge which spins the tubes of homogenate at progressively high speeds to create a centrifugal force

Question 29

In cell fractionation, what is the process of ultracentrifugation?

Answer 29

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