Electron Microscopy

Question 1

Define resolution

Answer 1

the ability to distinguish between two very closely positioned objects

Question 2

Why is resolution important?

Answer 2

because magnification alone does not make an image clearer, it just makes it bigger

resolution is required to make an image clear

Question 3

What is the maximum resolution of a light microscope? What does this mean?

Answer 3

0.2 micrometers with the 100x objective lens/total 1000x magnification

There must be at least 0.2 micrometers between the two spots to be able to see them as separate objects with a light microscope

Question 4

What is the formula for resolution? what does each variable stand for?

Answer 4

D = 0.61 lambda/ n sin alpha

D = the minimum distance between two distinguishable objects

lambda = wavelength of light used

n = refractive index of medium between specimen and objective lens

alpha = angular aperture (half angle) of the cone of light entering the objective lens from the specimen

Question 5

Why does resolution increase so dramatically with electron microscopes?

Answer 5

the shorter the wavelength, the better the resolution

electron microscopes use electrons as an energy source instead of light

electrons have shorter wavelengths than light

Question 6

Which has a higher resolving power: electron or light microscope?

Answer 6

electron

Question 7

Describe electron microscopes

Answer 7

Use an electron beam to pass through the specimen

Question 8

What are the two kinds of electron microscopy?

Answer 8

Transmission EM

Scanning EM

Question 9

Describe transmission EM

Answer 9

Forms images as a 2D slice of an object by using electrons that are TRANSMITTED through a specimen

Question 10

What is the range of resolution for TEM?

Answer 10

3-5 Å = 0.00003-0.00005 micrometers

Question 11

Describe scanning EM

Answer 11

Forms 3D images of an object by using electrons that are BOUNCED OFF the surface of a specimen

Question 12

What is the range of resolution for SEM?

Answer 12

5-10 nm

Question 13

What size of specimen would you use in a SEM?

Answer 13

larger sized specimens

Question 14

T or F: With both SEM and TEM, you can see the internal organelles of a specimen

Answer 14

FALSE.

Only with TEM could you see the internal organelles of a specimen because the electrons transmit through it

You could not see with the SEM because the electrons bounce off the surface of the specimen

Question 15

Describe the structure of an EM and how an EM works?

Answer 15

Top of column contains a cathode which is heated to provide a source of electrons

a vacuum is created inside the EM in order to direct the electrons downwards and reduce scattering

The beam of electrons is focused by electromagnetic lenses in the wall of the column

the specimen is on a small metal grid that is inserted into a grid holder which is inserted into the column of the EM

Question 16

What is the purpose of creating a vacuum in EM?

Answer 16

In order to direct the electrons downwards toward the specimen

without the vacuum, the electrons would scatter by collision with gas molecules

Question 17

What does it mean if spots on the image produced by an EM of the specimen are darker than others?

Answer 17

Fewer electrons hit that spot because the specimen was denser there

Question 18

Why will electrons not hit the denser parts of a specimen?

Answer 18

electrons may reflect and scatter after hitting specimen

the greater number of specimen atoms, the greater the probability of electrons scattering after hitting the specimen

Question 19

What can be used in EM to create better images? Why is this necessary?

Answer 19

Heavy metal stains are used to coat specimen in order to increase scatter and contrast because common cell atoms (O, H, etc) do not scatter electrons well

Question 20

Why is scattering important in EM?

Answer 20

It creates contrast between light and dark in the images produced because they are not in colour

Question 21

How do tissues need to be prepared for using an EM?

Answer 21

dehydrated, embedded in wax/plastic medium and sliced VERY VERY VERY THINLY

Question 22

Give an example of when you would use a TEM?

Answer 22

If you wanted to see the cristae in a mitochondria

Question 23

Give an example of when you would use an SEM?

Answer 23

If you wanted to see the shape of something

Question 24

In TEM what can be done to provide extra contrast?

Answer 24

negative staining coats the grid with a substance to remove holes and drops of heavy metal stain added across the whole grid so that the specimen stands out better

Question 25

In what situations would you use negative staining in TEM?

Answer 25

for small particulates like ribosomes, viruses, multi-subunit enzymes

Question 26

How does freeze fracturing relate to EM?

Answer 26

You can look at freeze fractured membranes under EM and use TEM

Question 27

What is the goal of SEM?

Answer 27

to produce an object that has the same shape and surface properties as the living state but has no fluid so it can be looked at under a vacuum

removing all water from living cells without disrupting cell structure

Question 28

How is the water from living cells removed without disrupting cell structure in SEM?

Answer 28

water replaced with liquid transitional fluid (CO2) at specific temperature and pressure

dried specimen coated with gold –> suitable target for electron beam

e scatter to make a 3D image