2.1 - Microscopy

Question 1

What is Magnification?

Answer 1

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

Magnification = size of the image = size of the real object

Question 2

What is resolution? What is it determined by?

Answer 2

The minimum distance between two objects in which they can still be viewed as separate objects

Higher resolution = clearer image.

This is determined by the wavelength of light in a light microscope and by the wavelength of the electron beam in an electron microscope.

Question 3

What is the formula to calculate magnification?

Answer 3

Magnification = Image size ÷ object size

Question 4

What is the eyepiece graticule?

Answer 4

A small piece of glass with a measurement scale etched on its surface that fits inside a microscope eyepiece.

Question 5

What is the stage micrometer? What is each division equal to? What is used for?

Answer 5

A microscope slide that has a ruler/scaled bar etched into it.

The scale is usually 1mm long and has 100 divisions so each division in 10µm.

It is used to calibrate the eyepiece graticule.

Question 6

How do you calibrate the eyepiece graticule?

Answer 6

1. Line up the stage micrometer and eyepiece graticule whilst looking through the eyepiece.
2. Count how many divisions on the eyepiece graticule fit into one division on the micrometer scale.
3. Each division on the micrometer is 10µm, so this can be used to calculate what one division on the eyepiece graticule is at that current magnification.

Question 7

What are the 6 key features of scientific drawings?

Answer 7

1. Draw in Pencil.
2. Title the diagram to indicate what the specimen is.
3. State the magnification that you are drawing it from.
4. Annotate cell components, cells and sections of tissue visible.
5. Do no sketch - only use solid lines that do not overlap.
6. Do not colour in or shade.

Question 8

What is differential staining?

Answer 8

A techniques involving many differential chemical stains being used to stain different parts of a cell in different colours.
E.g., crystal violet and acetic orcein.

Question 9

What are the 2 types of microscopes? Give examples.

Answer 9

1. Light (Optical) Microscope.
2. Electron Microscope:
   A) Transmission Electron Microscope (TEM)
   B) Scanning Electron Microscope (SEM)

Question 10

What are the advantages of using a light microscope? (5)

Answer 10

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

Question 11

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

Answer 11

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

Question 12

Describe how a transmission electron microscope (TEM) works

Answer 12

1. The sample must be thin and stained.
2. Pass a high-energy beam of electrons through a thin slice of specimen.
3. More dense structures appear darker since they absorb more electrons.
4. Focus image onto fluorescent screen or photographic plate using electromagnets in a vacuum.

Question 13

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

Answer 13

1. High magnification (x1,500,000)
2. High resolution (0.5nm)
3. Provides detailed images of interior substances.

Question 14

Describe the disadvantages of using a Transmission Electron Microscope. (6)

Answer 14

1. Can only see dead specimen
2. Harsh chemicals used in preparation which can cause artefacts - complex staining method
3. Expensive
4. Training required + Vacuum required.
5. Black and white images (false colouring).
6. Extremely thin specimens required.

Question 15

What can you see with a TEM?

Answer 15

2D images of details within organelles.
For example:
* cristae in the mitochondria
* grana in the chloroplasts

Question 16

Describe how a scanning electron microscope (SEM) works.

Answer 16

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

Question 17

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

Answer 17

1. High magnification (x1,500,000)
2. High resolution (5nm)
3. Can see details of the surfaces of structures
4. Produces a 3D image.

Question 18

Describe the disadvantages of using a Scanning Electron Microscope (SEM). (6)

Answer 18

1. Can only see dead material
2. Harsh chemicals used in preparation which can cause artefacts - complex staining methods.
3. Expensive
4. Black and white image (false colouring).
5. Thick sample is unusable - sample has to be very thin.
6. Vacuum required.

Question 19

What can you see with a SEM?

Answer 19

3D image of the surface of cells and organelles.

Question 20

Describe how a laser scanning confocal microscope works.

Answer 20

The image is created as teh microscope scans the specimen point-by-point using a focused laser beam.

Can see a 2D or 3D image.

Question 21

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

Answer 21

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 22

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

Answer 22

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

Question 23

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

Answer 23

1. Objective lens magnifies the specimen
2. Eyepiece lens magnifies image (from objective lens)
3. Higher magnification (produced than with just one lens)

Question 24

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

Answer 24

Magnification = x2000
Resolution = 200 nm

Question 25

What is the magnification and resolution of a TEM?

Answer 25

Magnification = x1,500,000 Resolution = 0.5 nm

Question 26

What is the magnification and resolution of a SEM?

Answer 26

Magnification = x1,500,000 Resolution = 5nm