3.2.1.5 - Light and Electron Microscopes

Question 1

What you see when looking through a microscope is called the

Answer 1

Image

Question 2

What are the disadvantages of a light microscope?

Answer 2

• Low resolution due to ‘longer’ wavelength of light.
• Low magnification (X1,250 max)
• Thin specimens may not represent true specimen.

Question 3

What are the advantages of a light microscope?

Answer 3

• Easy to use (no special training required)
• Cheap (<£1 K)
• True colour images but may sometimes require staining.
• Can observe live specimens

Question 4

Define microscope resolving power.

Answer 4

The ability of a microscope to differentiate between 2 close together objects.

Question 5

What is another term for resolution?

Answer 5

Resolving power

Question 6

What is meant by magnification?

Answer 6

How much bigger an object looks under a microscope.

Magnification = Image Size ÷ Actual Size

Question 7

What are the advantages of a transmission electron microscope (TEM)?

Answer 7

• High resolving power (0.1 nm)
• High magnification (X500, 000)
• Provides detailed images of internal structures of cells.

Question 8

Name the 3 main microscopes used by scientists.

Answer 8

1. Light microscope
2. Scanning electron microscope (SEM)
3. Transmission electron microscope (TEM)

Question 9

What are the advantages of a scanning electron microscope?

Answer 9

• High resolution (20 nm)
• High magnification (X200, 000)
• 3D images

Question 10

Why do electron microscopes have a greater resolving power than light microscopes?

Answer 10

• They use electrons to interact with the specimen.
• Electrons have a shorter wavelength so interact more witht he specimen.

Question 11

What are the disadvantages of a transmission election microscope (TEM)?

Answer 11

• Special training is required before use.
• Samples must be dead as electrons are fired through a vacuum and stains containing heavy elements are used.
• ‘Artefacts’ can be present in image from staining process.
• Sample must be 1 cell thick to allow electrons to penetrate specimen.
• Black and white images only so false colour must be used.
• 2D images - 3D possible but complicated and slower than SEM.
• High cost
• High energy electron beams can destroy the specimen.

Question 12

What is the resolving power of a light microscope and what does this mean?

Answer 12

2 µm

It can differentiate between objects up to that distance apart.

Question 13

What are the disadvantages of a scanning electron microscope (SEM)?

Answer 13

• Special training is required before use.
• Samples must be dead as electrons are fired through a vacuum and stains containing heavy elements are used.
• ‘Artefacts’ can be present in image from staining process.
• Black and white images only so false colour must be used.
• Cannot see inside specimens.
• High cost
• High energy electron beams can destroy the specimen.

Question 14

What are the main differences between scanning and transmission electron microscopes?

Answer 14

Question 15

Does light or electons have the shortest wavelength?

Answer 15

Electrons

Question 16

What is cell fracitonation?

Answer 16

The process by which cells are broken up and organelles are separated out.

Question 17

Describe the stages of cell fractionation.

Answer 17

1. Tissue is placed in a cold, buffered, isotonic solution.
2. Tissue and cells are broken up using a homogeniser (blender)
3. Homogenate is filtered to remove cell debris.
4. Nuclei in the homogenate are separated by being spun at low speed using a centrifuge (ultracentrifugation)
5. Supernatent is removed leaving pellet of nuclei.
6. Supernatent spun at medium speed to create pellet of mitochondrion.
7. Supernatent removed and spun at high speed to create pellet of ribosomes.

Question 18

Before cell fractionation can take place, the tissue to be observed is placed in a cold, buffered, isotonic solution. Why is the solution cold?

Answer 18

To reduce enzyme activity within the cell that could break down organelles.

Question 19

Before cell fractionation can take place, the tissue to be observed is placed in a cold, buffered, isotonic solution. Why is the solution isotonic?

Answer 19

If the solution was not of the same water potential as the tissue then organelles could burst as a result of osmotic gain or loss of water.

Question 20

Before cell fractionation can take place, the tissue to be observed is placed in a cold, buffered, isotonic solution. Why is the solution buffered?

Answer 20

So that pH is maintained.

A change in pH could affect the enzymes within the cells.

A change in pH could affect the structure of organelles within the cells.

Question 21

What is a homogeniser?

Answer 21

A blender used to break up tissues and cells and release organelles.

Question 22

What is a homogenate?

Answer 22

The resulting fluid after homogenisation

Question 23

What is a centrifuge?

Answer 23

A machine that spins tubes of homogenate at varying speeds (used in cell fractionation)

Question 24

name 2 organelles foundin eukaryotes that cant be seen with a light microscope

Answer 24

mitochondria, ribosome, ER, cell surface membrane

Question 25

when preparing a slide for viewing why must you press down hard on the coverslip (without breaking!)

Answer 25

To squash tissue (dont push sideways - avoid cells rolling together)

Question 26

why should the specimen be thin?

Answer 26

single layer of cells

to allow light to pass through

Question 27

how do you prepare a temporary mount for viewing?

Answer 27

add drop of water to slide

take a thin section of tissue - 1 cell thick

place on glass slide (float on water)

add stain

place on coverslip

press down firmly

Question 28

Contrast a TEM and a Optical microcope

Answer 28

TEM electrons - light optical

TEM greatER resolution

TEM can see smallER organelles e.g. ribosomes

TEM only dead specimens - light can view dead and live

TEM no colour - optical can

TEM needs thinnER specimen

Question 29

If doing a scientific drawing at an image under the microscope what should you do to ensure the quality of it?

Answer 29

dont use shading

use SINGLE lines (no sketching)

add labels/annotations

dont cross label lines

add a magnification/scale bar

Question 30

What type of image does an SEM provide?

Answer 30

3D

Question 31

How caould you determine the mean length of a cell using an eye piece graticule?

Answer 31

calibrate the graticule using a ruler/stage micrometer

Measure the length of a number of cells (at random) using the graticule

calculate a mean

Question 32

Give some top tips when making a biological drawing

Answer 32

1. continuous lines - no sketching
2. no shading
3. draw what you see
4. add a scale bar or state magnification/scale
5. Add labels