2.1.1 Microscopy

Question 1

What are the 4 types of microscope?

Answer 1

• Light microscope
• transmission electron microscope [TEM]
• scanning electron microscope [SEM]
• laser scanning confocal microscope (type of light microscope)

Question 2

How does a Scanning Electron microscope work?

Answer 2

A beam of elections is sent across the surface of the specimen and the reflected electrons are collected to form the image

Question 3

Does a scanning electron microscope produce a 2D or 3D image?

Answer 3

SEM produces a 3D image

Question 4

Does a SEM give a higher or lower resolution than a TEM

Answer 4

SEM produces lower resolution images than TEM

Question 5

How does a Transmission electron microscope work?

Answer 5

Use electromagnets, a beam of electrons pass through the specimen and focussed to produce the image.
Denser parts of the specimen absorb more electrons, so they look darker.

Question 6

Do transmission electron microscopes have a higher or lower resolution than SEM

Answer 6

Transmission electron microscopes have a very high resolution

Question 7

How does a laser scanning confocal microscope work?

Answer 7

• Use laser beams,
• A single spot of light is moved across the sample (point illumination).
• Specimen is dyed with fluorescent dye
• Laser causes dyed parts to fluorescence - give off light
• light filtered through pinhole onto detector
• pinhole means out of focus light is blocked, producing a clear image.

Question 8

Does a laser scanning confocal microscope produce a 3D or 2d image

Answer 8

Laser scanning confocal microscope images can be both 2D and 3D

Question 9

Properties of light microscope:
Uses…
Magnification
Resolution
Dead / alive specimen

Answer 9

Light microscope:
Uses light radiation and glass lenses to focus
Magnification x1500 - x2000
Resolution 200nm / 0.2um
No vacuum - specimens can be alive/dead
Stained with eyes
Viewed with eye
View whole cells or tissues

Question 10

Properties of scanning electron microscope:
Uses…
Magnification
Resolution
Dead / alive specimen
Stained with…
Viewed with…
Image colour…

Answer 10

• Uses electrons from electron gun, focused by electromagnets
• magnification x100 000 -x500 000
• resolution 3-10nm / 0.002um
• need vacuum - dead specimen
• stained with heavy metals
•Viewed on computer
• B&W

Question 11

Properties of transmission electron microscope:
Uses…
Magnification
Resolution
Dead / alive specimen
Stained with…
Viewed with…
Image colour…

Answer 11

Uses electron from electron gun, focussed by electromagnets
•Magnification - x500 000 - x2000 000
•Resolution - 0.2 - 0.5nm / 0.0002um
• need vacuum - dead specimen
• stained with heavy metals
• Viewed on computer
• B&W

Question 12

Features of light microscope images

Answer 12

• Light background
• 2D
• Variety of colours
• Show details larger than 200nm only
• Large organelles only - (nuclei, vacuole, cholroplasts, mitochondria) / parts of organisms/whole cells
• observing small organisms

Question 13

Features of laser scanning confocal microscope

Answer 13

• 2D computer can make 3D
• black background
• limited colour
• observing stained structures within cells/whole cells

Question 14

Features of transmission electron microscope

Answer 14

• Always 2D
• only grayscale unless false colour
• v high magnification
• high resolution - grainy image
• observing organelles within cells or whole cells

Question 15

Features of scanning electron microscope

Answer 15

• 3D (inside and outside)
• outside surfaces
• greyscale/ false colour

Question 16

Identify the type of microscope used when shown a photomicrograph

Answer 16

Light: light background
TEM: false colour, v high resolution
SEM: black background, false colour, 3D looking, v high resolution
Laser scanning confocal: black black ground, limited colour

Question 17

Timeline showing development of microscope

Answer 17

Romans experiment with glass lenses

13th century invention of spectacles

15th century century telescope

1609 Galileo Galilei first ‘thing’ to be called microscope (compound light)

Question 18

How did the development of the microscope intifuence scientific thinking.

Answer 18

Allowed cells to be observed - disproved spontaneous generation
Allowed theories to be presented and disproved then replaced when technology improves

Question 19

Describe and explain the characteristics of a good slide preparation

Answer 19

No artefacts
Thin
Transparent
Clean slides + coverslips
Good contrast
Cells spread out with limited overlap
Appropriate vowme of liquid

Question 20

Why do slide preparations need to be thin

Answer 20

To let light through
Individual cells can be identified

Question 21

Why is staining useful for light microscopy

Answer 21

Many specimens are transparent
Some parts of the object take up more dye
Creates a contrast in colour or intensity between objects and their surroundings

Different stains bind to different molecules

Question 22

What is a polychromatic stain?

Answer 22

Polychromatic stains show different colours with different molecules

Question 23

Can more than one stain be used at once?

Answer 23

Yes, because they bind to different molecules

Question 24

Give 4 examples of stains

Answer 24

Methylene blue
eosin
Toluidine blue
Iodine in potassium iodide

Question 25

What do eosin, methylene blue, Toluidine blue
Iodine in potassium iodide stain ?

Answer 25

Methylene blue - DNA
Eosin- cell cytoplasm

• Toluidine blue - phloem - red, xylem - blue, DNA-blue
• Iodine in potassium iodide - starch blue / black

Question 26

What properties does a stain need to have to be use for light microscopy?

Answer 26

Differential staining - stain some objects and not others
↳ contrast visible
Intense colour - v. Small amount of biological material but has to be visible

Question 27

What is the formula for magnification ?

Answer 27

Magnification = image size / actual size

Question 28

How to convert from
mm —> um —> nm

Answer 28

mm —> um —> nm
x1000 x1000

Question 29

Define resolution

Answer 29

The ability to distinguish between 2 separate points

Question 30

Define magnification

Answer 30

Number of times larger the image is compared to the object

Question 31

What is the difference between magnification and resolution?

Answer 31

Magnification is enlargement not always increased detail
Resolution is distance
Magnification has no units

Question 32

Resolution and max magnification of all the microscopes

Answer 32

Light : 200nm and 1500-2000x
SEM: 3-10nm and 100,000 - 500,000x
TEM: 0.2nm and 500,000 - 2,000,000