2.2 Microscopy

Question 1

Name the 3 types of microscope?

Answer 1

Optical, Electron (Transmission Electron Microscope, Scanning Electron Microscope)

Question 2

Magnification Formula:

Answer 2

magnification = size of image / size of real object

Question 3

Magnification is …

Answer 3

how many times bigger the image produced by the microscope is than the real-life object you are viewing

Question 4

Resolution is …

Answer 4

the ability to distinguish between objects that are close together

Question 5

Optical microscopes have a … (1) resolution, because visible light has … (2)

Answer 5

(1) low
(2) long wavelengths

Question 6

How does an optical microscope produce an image?

Answer 6

Uses convex lenses to magnify images of small samples with visible light.

Question 7

Advantages of Transmission Electron Microscope

Answer 7

• very high resolution images
• allows the internal structures within cells to be seen

Question 7

How does an electron microscope produce an image?

Answer 7

Uses beams of electrons to form an image.
TEMS: Use electromagnets to focus a beam of electrons on the sample, this beam of electrons is transmitted through the specimen. Denser parts of the specimen absorb more electrons making these parts appear darker on the final image produced.
SEMS: Scan a beam of electrons across the specimen. This beam bounces off the surface of the specimen and the electrons are detected, forming an image.

Question 8

Disadvantages of Transmission Electron Micrscope

Answer 8

• can only be used with very thin specimens
• can not observe live specimens
• lengthy treatment required to prepare specimens means that artefacts can be introduced
• do not produce a colour image

Question 9

Advantages of Scanning Electron Microscope

Answer 9

• can be used on thick specimen
• allow the external, 3D structure of specimens to be observed

Question 10

Disadvantages of Scanning Electron Microscope

Answer 10

• lower resolution than TEM
• can not observe live specimens
• do not produce a colour image

Question 11

Optical vs Electron (size)

Answer 11

Optical: Small and portable
Electron: Large and installation means it can’t be moved

Question 12

Optical vs Electron (magnification + resolution)

Answer 12

Optical: mag → x2000 res → 200nm
Electron: mag → >x500,000 res → 0.5nm (larger and higher)

Question 13

Optical vs Electron (specimens)

Answer 13

Optical: Live
Electron: Dead

Question 14

Optical vs Electron (preparation)

Answer 14

Optical: Simple
Electron: Complicated

Question 15

Optical vs Electron (vacuum)

Answer 15

Optical: ✗
Electron: ✓

Question 16

What is cell fractionation?

Answer 16

Separating cell organelles from each other.

Question 17

What are the steps of cell fractionation?

Answer 17

1. Homogenisation
2. Filtration
3. Ultracentrifugation

Question 18

What is homogenisation?

Answer 18

The biological term for the breaking up of cells.

Question 19

The homogenate solution must be …

Answer 19

Ice Cold
Buffered
Isotonic

Question 20

Ice Cold?

Answer 20

To reduce the activity of enzymes that break down organelles.

Question 21

Buffered?

Answer 21

To prevent organelle proteins from denaturing.

Question 22

Isotonic?

Answer 22

So water potential is not affected to prevent water moving into organelles by osmosis which could lead to cell damage.

Question 23

What does the homogeniser do?

Answer 23

This breaks the plasma membrane of the cells and releases the organelles into a solution called the homogenate.

Question 24

Why is the homogenate filtered?

Answer 24

To remove any large cell debris that were not broken up.

Question 25

What is ultracentrifugation?

Answer 25

The filtrate is placed into a tube and the tube is placed in a centrifuge.
Then the filtrate is spun at increasing speeds to separate the different organelles.

Question 26

Order of mass of the organelles.

Answer 26

1. Nuclei
2. Chloroplasts (for plant tissues)
3. Mitochondria
4. Lysosomes
5. Endoplasmic reticulum
6. Ribosomes

Question 27

In what order are the organelles separated.

Answer 27

Heaviest organelles (nucleus/mitochondria) first at the slowest speeds. Lighter organelles last at faster speeds.

Question 28

Process of ultracentrifugation.

Answer 28

Spinning causes the largest, heaviest organelles to settle at the bottom of the tube, where they form a thick sediment known as a pellet. The rest of the organelles stay suspended in the solution above the pellet, known as the supernatant.
This supernatant is re-spun at a higher speed.

Question 29

Process of ultracentrifugation.

Answer 29

Spinning causes the largest, heaviest organelles to settle at the bottom of the tube, where they form a thick sediment known as a pellet. The rest of the organelles stay suspended in the solution above the pellet, known as the supernatant.
This supernatant is re-spun at a higher speed.