2 - MZH - Light and electron microscopy

Question 1

Define magnification

Answer 1

The degree to which the size of an image is larger than the object itself.

Greater magnification = larger image

Question 2

Define resolution

Answer 2

Degree of which it is possible to distinguish between 2 objects that are very close together.

Greater resolution = more detail

Question 3

Why does using blue light with a light microscope improve resolution?

Answer 3

Blue light has a shorter wavelength.

The shorter the wavelength the better the resolution

Question 4

TEM?

What can you see?

Answer 4

Transmission electron microscope

A cross section of the object is seen since the electrons have passed through the specimen

Question 5

SEM?

What can you see?

Answer 5

Scanning electron microscope

The surface of the object is seen since electrons have been reflected from the specimen

Question 6

LM and TEM

Form of radiation used?

Answer 6

LM = light

TEM = Electrons (electron beams)

Question 7

LM and TEM

Radiation wavelength (longer/shorter)?

Answer 7

LM = longer

TEM = shorter

Question 8

LM, TEM, SEM

Limit of resolution?

Answer 8

LM = 0.2µm (or 200 nm)

TEM = 0.0001µm (or 0.2-0.5 nm)

SEM = 3-10 nm

Question 9

LM, TEM, SEM

Max useful magnification?

Answer 9

LM = x 1500 - 2000

TEM = x 500,000 - 2,000,000

SEM = x 100,000 - 500,000

Question 10

LM and TEM

Form of lens used?

Answer 10

LM = Glass

TEM = Electromagnets

Question 11

LM and TEM

Section thickness (thicker/thinner)?

Answer 11

LM = Thinner

TEM = Even thinner than LM

Question 12

LM and TEM

Types of stains used?

Answer 12

LM = Coloured e.g. Methylene blue

TEM = Heavy metals e.g. Lead

Question 13

LM and TEM

Specimens living or dead?

Answer 13

LM = Living & dead

TEM = Dead only

Question 14

Purpose of sectioning?

Answer 14

So that light can pass through it

Question 15

Purpose of staining?

Answer 15

Improve contrast

Question 16

Name the steps of the preparation for light microscopy (7)

Answer 16

1 - Fixation

2 - Dehydration

3 - Clearing

4 - Embedding

5 - Sectioning

6 - Staining

7 - Mounting

Question 17

What does Methylene blue do?

Answer 17

Stains nuclei blue (used with a LM)

Question 18

Equation for magnification = ?

Answer 18

Magnification = image length / actual length (IAM triangle)

Question 19

1mm = m?

1mm = µm?

1mm = nm?

1µm = nm?

Answer 19

1mm = 1 x 10m^-3

1mm = 1 x 10µm³

1mm = 1 x 10nm⁶

1µm = 1 x 10nm³

m → mm → µm → nm

Question 20

Advantages of electron microscopes (4)

Answer 20

• Higher resolution.
• More detailed images of the structures inside cells.
• SEM produced a final 3D image. Not possible for LM.
• Laser confocal microscopes don’t damage tissue so they can be used to screen inside of the eye to diagnose eye conditions.

Question 21

Limitations of electron microscopes (4)

Answer 21

• Expensive
• Preperation of specimen is complex (requries special training)
• Image produced is in black/white (unless you use a computer to generate a false colour image)
• View DEAD specimens only

Question 22

Advantages of light/ optical microscope (3)

Answer 22

• Easy to use (no special training required)
• Easy to prepare specimen. Unlikely to distort it during preperation.
• Coloured images produced

Question 23

Limitation of light/ optical microscope (2)

Answer 23

Lower magnification

Lower resolution

Question 24

Why do you use blue light when using a LM?

Answer 24

Blue light has a shorter wavelength than visible light