Microscopy

Question 1

What is the difference between resolution and magnification?

Answer 1

• Resolution is the ability to distinguish between 2 points -> if you cannot resolve 2 points, you see them as one
• Increasing the size of an image but does not increase the resolution
• The object will appear larger but just as blurred

Question 2

What are the types of microscope ?

Answer 2

• Optic microscope
• Transmission electron microscope
• Scanning electron microscope

Question 3

How do light microscopes work?

Answer 3

• Specimens are illuminated with light
• The light is focused using glass lenses and viewed using the eye. All light microscopes today are compound microscopes, which means they use several lenses to obtain high magnification.

Question 4

What are the advantages and disadvantages of light microscopes?

Answer 4

• Poor resolution because of relatively long wavelength of light. The resolution is 200nm so anything closer then that will only be seen as one point.
• Only 2D slice can be observed
• Specimens can be living or dead
• You can see colour

Question 5

How do TEM microscopes work?

Answer 5

• Electron beam passes through a thin section of a specimen,
• Parts of the specimen absorb electrons and therefore appear dark, other parts of the specimen allow the electrons to pass through and so appear bright.

Question 6

What are the advantages and disadvantages of using TEM ?

Answer 6

• Specimens must be thin
• Specimens must be dead as they must be in a vacuum so that the electrons can pass through.
• An image that can be photographed
  produced on a screen
• Black and white
• 2D slice
• Really high resolution because of the shorter wavelength of electrons (0.1nnm)
• Can magnify up to 500,000 times

Question 7

What are the advantages and disadvantages of using TEM ?

Answer 7

• Specimens must be thin
• Specimens must be dead as they must be in a vacuum so that the electrons can pass through.
• An image that can be photographed
  is produced on a screen
• A 2D image is produced
• a black and white image is produced
  — Can resolve 0.1nm and can magnify up to 500,000 times

Question 8

How does SEM work?

Answer 8

• SEM directs a beam of electrons on the surface of the specimen and the beam is passed back and fourth across the specimen. The electrons are scattered and detected.

Question 9

What are the advantages and disadvantages of SEM?

Answer 9

• All the limitations of the TEM also apply to the SEM except that the specimen doesn’t need to be really thin
• 3D image can be generated. Allows detailed study of surfaces.
• Lower resolving power than TEM
• Resolving power of around 20nm

Question 10

Equation for magnification

Answer 10

Magnification = image size / actual size

Question 11

Compare the units used in microscopy to a meter

Answer 11

• Millimeter (mm) -> 1/1000
• Micrometer (um) -> 1/1,000,000
• Nanometer (nm) -> 1/1,000,000,000
• Picometer (pm) -> 1/1,000,000,000,000

Question 12

How to measure the actual size of a specimen using a microscope ?

Answer 12

• Use an eyepiece graticule and stage micrometer
• Before you can use the graticule it must be calibrated.
• To do this you line up the scale on the eyepiece with that of the micrometer using the objective lense .
• For example, you may use the 400x objective lens that magnifies 400 times.
• Suppose this shows that 50 graticule units are equivalent to 10 micrometer units.
• If each micrometer unit is 10um then each micrometer unit equals 2 um.
• If an objective lens magnifying 100 times is then used, each graticule unit would be equivalent to 8um.

Question 13

Rules for drawing low power drawings

Answer 13

• Identify the different tissue, using high power to help if necessary
• Draw all tissue and completely enclose each tissue by lines
• Don’t draw individual cells
• Accuracy is important- the specimen will not necessarily look like a textbook drawing
• A representative portion may be drawn if the structure if the structure is symmetrical

Question 14

Rules for drawing high power drawings

Answer 14

• Draw only a few representative adjacent cells. If all the cells are similar, then three cells is often sufficient to show cell structure and how cells are arranged in relation to each other
• Don’t shade

Question 15

Steps of differential centrifugation

Answer 15

• Tissue is cut up and kept in a cold, isotonic buffered solution
• Cut up tissue further broken down in a homogeniser
• Homogenised tissue is spun in an ultracentrifuge at a low speed for 10 minutes
• Filtrate spun at different speeds to form different pellets

Question 16

Explain the first step in differential centrifugation

Answer 16

• Cold -> reduce enzyme activity so organelles aren’t digested
• Isotonic -> prevent organelles bursting or shrinking as a result of the osmotic gain or loss of water
• Buffered -> maintain constant PH

Question 17

Explain the second step in differential centrifugation

Answer 17

• Cells broken up
• Organelles released from the cells membrane
  -resultant fluid known as the filtrate or homogenate is formed
• It is filtered to remove unwanted large pieces of debris

Question 18

Explain the third step in differential centrifugation

Answer 18

• The third step is ultracentrifugation
• The filtrate is spun at very high speeds to separate organelles. The centrifuge force separates organelles into densities

Question 19

What is done with the result of ultracentrifugation

Answer 19

• Densest organelle forced to the bottom
• Thin sediment or pellet is formed
• Fluid at the top (supernatant) is removed

Question 20

What is the order of the pellets collected

Answer 20

• At the lowest speed the densest organelles are collected. This pellet includes nuclei and cellular debris
• At the medium speeds decreasingly dense organelles are collected. This pellet includes mitochondria and chloroplasts
• At the highest speed the least dense of organelles are collected. This pellet includes ribosomes.