Biology Topic 2.1.1 - Microscopy (Miscroscopes)

Question 1

What is resolution?

Answer 1

Resolution is ability to distinguish two close points as separate / to see finer level of detail
Higher resolution = clearer image.

Question 2

What is magnification?

Answer 2

Factor by which the image is larger than the actual specimen.

Question 3

What is the formula to calculate magnification?

Answer 3

Magnification = Image size ÷ object size

Question 4

Outline how a student could prepare a temporary mount of tissues for a light microscope.

Answer 4

1. Obtain thin section of tissue (e.g., using ultratome or by maceration).
2. Use a pipette to place a small drop of water onto the centre of the glass slide.
3. Place plant tissue in a drop of a water, using forceps.
4. Stain tissue on a silde to make structures visible.
5. Add coverslip using a mounting needle at 45° to avoid trapping air bubbles.

Question 5

Describe how light microscopes work.

Answer 5

1. Lenses focus rays of light and magnify the view of a thin slice of specimen.
2. Different structures absorb different amounts and wavelengths of light.
3. Reflected light is transmitted to the observed via the objective lens and eyepiece.

Question 6

What are the advantages of using a light microscope? (4)

Answer 6

1. Whole cells and tissues can be seen
2. Easy sample preparation
3. Cheap to buy (<£1k)
4. can use live specimens.

Question 7

What are the disadvantages of using a light microscope? (3)

Answer 7

1. Low magnification (x1500)
2. Low resolution (0.2µm)
3. Specimens are thin = may not be representative + cannot see all organelles.

Question 8

Describe how a transmission electron microscope (TEM) works

Answer 8

1. Pass a high-energy beam of electrons through a thin slice of specimen.
2. More dense structures appear darker since they absorb more electrons.
3. Focus image onto fluorescent screen or photographic plate using magnetic lenses.

Question 9

Describe the advantages of using a Transmission Electron Microscope. (3)

Answer 9

1. High magnification (up to 5000 000 times)
2. High resolution
3. Provides detailed images of interior substances.

Question 10

Describe the disadvantages of using a Transmission Electron Microscope. (5)

Answer 10

1. Can only see dead material
2. Harsh chemicals used in preparation which can cause artefacts
3. Expensive
4. Training required.
5. Black and white images (false colouring)

Question 11

Describe how a scanning electron microscope (SEM) works.

Answer 11

1. Focus a beam of electrons onto a specimen’s surface using electromagnetic lenses.
   2, Reflected electrons hit a collecting device and are amplified to produce an image on a photographic plate.

Question 12

Describe the advantages of using a Scanning Electron Microscope (SEM). (4)

Answer 12

1. High magnification (up to 5000 000 times)
2. High resolution
3. Can see details of the surfaces of structures
4. Produces a 3D image.

Question 13

Describe the disadvantages of using a Scanning Electron Microscope (SEM). (4)

Answer 13

1. Can only see dead material
2. Harsh chemicals used in preparation which can cause artefacts
3. Expensive
4. Black and white image (false colouring).

Question 14

Describe how a laser scanning confocal microscope works.

Answer 14

1. Focus a laser beam onto a small area on a sample’s surface using objective lenses.
2. Fluorophores in the sample emit photons.
3. A photomultiplier tube amplifies the signal onto a detector. An image is produced pixel by pixel in the correct order.

Question 15

What are the advantages of using a laser scanning confocal microscope? (4)

Answer 15

1. Can see living cells.
2. Can observe cell processes by tracking molecules.
3. Higher resolution than light microscopes (0.02µm).
4. Higher magnification (100,000x)

Question 16

What are the disadvantages of using a laser-scanning confocal microscope? (2)

Answer 16

1. Cannot see deep into cell tissue as light cannot penetrate.
2. More expensive and complex than a light microscope.

Question 17

What is the benefit of having two lenses - objective & eyepiece?

Answer 17

objective lens magnifies the specimen
eyepiece lens magnifies image (from objective lens)
higher magnification (produced than with just one lens)

Question 18

What is the magnification and resolution of a compound light microscope?

Answer 18

Magnification = x2000
Resolution = 200 nm

Question 19

What is the magnification and resolution of a TEM?

Answer 19

Magnification = x500,000
Resolution = 0.5 nm

Question 20

What is the magnification and resolution of a SEM?

Answer 20

Magnification = x500,000
Resolution = 3-10 nm

Question 21

How should you view a sample using a light microscope?

Answer 21

1. Start by clipping the slide containing the specimen you want to look at onto the stage.
2. Select the lowest-powered objective lens.
3. Use the coarse adjustment knob to bring the stage up to just below the objective lens.
4. Look down the eyepiece lens (which contains the ocular lens). Use the coarse adjustment knob to move the stage downwards, away from the objective lens until the image is roughly in focus.
5. Adjust the focus with the dine adjustment knob, until you get a clear image of what’s on the slide.

Question 22

What is the purpose of an eyepiece graticule and stage micrometre?

Answer 22

Sometimes you want to know the size of your specimen. And that’s where the eyepiece graticule and stage micrometre come in - they’re a bit like rulers.

Question 23

How do you set an eyepiece graticule and stage micrometer up on the light microscope?

Answer 23

1. An eyepiece graticule is fitted onto the eyepiece. It’s like a transparent ruler with numbers, but not units.
2. The stage micrometre is placed on the stage - it is a microscope slide with an accurate slide (it has units) and it’s used to work out the value of the divisions on the eyepiece graticule at a particular magnification.

This means that when you take the stage micrometre away and replace it with the slide containing your specimen, you’ll be able to measure the size of the specimen.