Electron Microscopy Flashcards
What is the range of light wavelength?
What is the resolution of light?
400-710 nm
Approx 200nm
What is the wavelength of electrons?
What is the resolution of electrons?
Approx 2.5pm
Approx 0.1nm
How do electron microscopes generally work?
Electrons emitted from filament
- -> accelerated by electric field (towards anode)
- -> beams focused by condenser lens (electromagnetic lens)
What conditions must be controlled in electron microscopes?
Vacuum
High pressure
What does the condenser lens do?
Focuses the electron beam
What is the difference between the way SEM + TEM work?
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
What are the 4 direct examination TEM techniques?
> Grids
Formvar
Contrast
Replication
What are the 5 TEM techniques for Sections from Tissues?
> Fixation > Dehydration > Embedding > Sectioning > Staining
Describe how samples are coated with formvar
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
How can sample contrast be enhanced?
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
What can be done to fragile samples?
Replicas made
- sample surface coated w/ film of evaporated carbon/plastic
then shadowed
Why is fixation needed?
Biological fine structure needs to be preserved during sample preparation
Why should small samples be used in fixation?
Chemicals must penetrate tissues to fix them
–> smaller sample
= rapid infiltration of fixative
What happens in dehydration?
10% - 100% ethanol series in 10% steps w/ gentle agitation
Describe the embedding process
- Place sample in BEEM capsule
- Infiltration w/ un-polymerised epoxy resin, Epon or Araldite
- Polymerisation of resin in BEEM capsule
- Remove resin block
Describe the sectioning process
- Ultra-microtome estimates section thickness by interference colours
- Sections picked up on formvar coated grids
What do fixatives vary in?
> Rate of penetration of tissues
Ability to fix different molecules
Shrinkage or swelling of tissues
What are the 4 ways of detecting cell component localisation in light microscopy?
> Histochemical dyes
Antibodies linked to FITC
GFP
Chromogenic enzyme substrate
What are the 4 techniques for detecting cell component localisation in electron microscopy?
> 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
What does peroxidase do?
Converts phenols to coloured electron dense products
What are the features of SEMs?
> Wide range of magnification(x10 to x100,000) > Great depth of focus > High resolution (<10nm) > Can rotate specimen > View large, irregular specimens
How must SEM samples be prepared?
Similar to TEM but w/ robust specimens
Delicate specimens need to be dehydrated - critical point drying
Describe the critical point drying process
- dehydrate in ethanol-water series
- replace w/ amyl acetate
- subject to liquid CO2 under pressure
- raise temp
- -> liquid CO2 converts to gas = leaves dry specimen
What are the problems with critical point drying?
What can be used to overcome these issues?
Shrinkage
Removal of substances soluble in organic solvents
Cryo-SEM
(Flash freeze in liquid N)
Why is Cryo-SEM good?
Sample retains water
No exposure to fixatives or solvents
Short preparation time
What are the 3 chemicals used during fixation?
Glutaraldehyde
Osmium tetroxide
KMNO4
