Electron Microscopy

Question 1

What is the range of light wavelength?

What is the resolution of light?

Answer 1

400-710 nm

Approx 200nm

Question 2

What is the wavelength of electrons?

What is the resolution of electrons?

Answer 2

Approx 2.5pm

Approx 0.1nm

Question 3

How do electron microscopes generally work?

Answer 3

Electrons emitted from filament

• -> accelerated by electric field (towards anode)
• -> beams focused by condenser lens (electromagnetic lens)

Question 4

What conditions must be controlled in electron microscopes?

Answer 4

Vacuum

High pressure

Question 5

What does the condenser lens do?

Answer 5

Focuses the electron beam

Question 6

What is the difference between the way SEM + TEM work?

Answer 6

TEM: electrons scatter or hit fluorescent screen at bottom of microscope
Specimen between 2 lenses

SEM: electrons focused onto metal coated specimen + scattered electrons from the metal are collected by detector
Specimen at end of 2 lenses

Question 7

What are the 4 direct examination TEM techniques?

Answer 7

> Grids
Formvar
Contrast
Replication

Question 8

What are the 5 TEM techniques for Sections from Tissues?

Answer 8

> Fixation 
> Dehydration 
> Embedding 
> Sectioning 
> Staining

Question 9

Describe how samples are coated with formvar

Answer 9

Formvar floats on surface of water in petri dish

• -> grids sitting on shit of wire glaze at bottom of dish lifted out of water
• -> formvar deposited on them

Question 10

How can sample contrast be enhanced?

Answer 10

Negative staining
- staining w/ heavy metals e.g lead, uranium, tungsten + gold

Shadowing

• heavy metal evaporated from wire in vacuum chamber
• -> casts shadow onto adjacent sample

Question 11

What can be done to fragile samples?

Answer 11

Replicas made
- sample surface coated w/ film of evaporated carbon/plastic
then shadowed

Question 12

Why is fixation needed?

Answer 12

Biological fine structure needs to be preserved during sample preparation

Question 13

Why should small samples be used in fixation?

Answer 13

Chemicals must penetrate tissues to fix them
–> smaller sample
= rapid infiltration of fixative

Question 14

What happens in dehydration?

Answer 14

10% - 100% ethanol series in 10% steps w/ gentle agitation

Question 15

Describe the embedding process

Answer 15

1. Place sample in BEEM capsule
2. Infiltration w/ un-polymerised epoxy resin, Epon or Araldite
3. Polymerisation of resin in BEEM capsule
4. Remove resin block

Question 16

Describe the sectioning process

Answer 16

1. Ultra-microtome estimates section thickness by interference colours
2. Sections picked up on formvar coated grids

Question 17

What do fixatives vary in?

Answer 17

> Rate of penetration of tissues
Ability to fix different molecules
Shrinkage or swelling of tissues

Question 18

What are the 4 ways of detecting cell component localisation in light microscopy?

Answer 18

> Histochemical dyes
Antibodies linked to FITC
GFP
Chromogenic enzyme substrate

Question 19

What are the 4 techniques for detecting cell component localisation in electron microscopy?

Answer 19

> Antibody linked to protein A-gold
- diff sizes can differentiate between 2 diff compounds simultaneously

> Enzyme localisation by linking product to heavy metal eg. lipases with Pb

> Enzyme localization if products are electron dense eg. peroxidase

Question 20

What does peroxidase do?

Answer 20

Converts phenols to coloured electron dense products

Question 21

What are the features of SEMs?

Answer 21

> Wide range of magnification(x10 to x100,000)
> Great depth of focus
> High resolution (<10nm)
> Can rotate specimen 
> View large, irregular specimens

Question 22

How must SEM samples be prepared?

Answer 22

Similar to TEM but w/ robust specimens

Delicate specimens need to be dehydrated - critical point drying

Question 23

Describe the critical point drying process

Answer 23

1. dehydrate in ethanol-water series
2. replace w/ amyl acetate
3. subject to liquid CO2 under pressure
4. raise temp
   - -> liquid CO2 converts to gas = leaves dry specimen

Question 24

What are the problems with critical point drying?

What can be used to overcome these issues?

Answer 24

Shrinkage
Removal of substances soluble in organic solvents

Cryo-SEM
(Flash freeze in liquid N)

Question 25

Why is Cryo-SEM good?

Answer 25

Sample retains water No exposure to fixatives or solvents Short preparation time

Question 26

What are the 3 chemicals used during fixation?

Answer 26

Glutaraldehyde Osmium tetroxide KMNO4