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Cell Biology > Electron Microscopy > Flashcards

Electron Microscopy Flashcards

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1
Q

What is the range of light wavelength?

What is the resolution of light?

A

400-710 nm

Approx 200nm

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2
Q

What is the wavelength of electrons?

What is the resolution of electrons?

A

Approx 2.5pm

Approx 0.1nm

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3
Q

How do electron microscopes generally work?

A

Electrons emitted from filament

  • -> accelerated by electric field (towards anode)
  • -> beams focused by condenser lens (electromagnetic lens)
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4
Q

What conditions must be controlled in electron microscopes?

A

Vacuum

High pressure

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5
Q

What does the condenser lens do?

A

Focuses the electron beam

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6
Q

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

A

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

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7
Q

What are the 4 direct examination TEM techniques?

A

> Grids
Formvar
Contrast
Replication

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8
Q

What are the 5 TEM techniques for Sections from Tissues?

A 
> Fixation 
> Dehydration 
> Embedding 
> Sectioning 
> Staining
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9
Q

Describe how samples are coated with formvar

A

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
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10
Q

How can sample contrast be enhanced?

A

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
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11
Q

What can be done to fragile samples?

A

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

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12
Q

Why is fixation needed?

A

Biological fine structure needs to be preserved during sample preparation

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13
Q

Why should small samples be used in fixation?

A

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

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14
Q

What happens in dehydration?

A

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

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15
Q

Describe the embedding process

A
  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
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16
Q

Describe the sectioning process

A
  1. Ultra-microtome estimates section thickness by interference colours
  2. Sections picked up on formvar coated grids
17
Q

What do fixatives vary in?

A

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

18
Q

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

A

> Histochemical dyes
Antibodies linked to FITC
GFP
Chromogenic enzyme substrate

19
Q

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

A

> 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

20
Q

What does peroxidase do?

A

Converts phenols to coloured electron dense products

21
Q

What are the features of SEMs?

A 
> Wide range of magnification(x10 to x100,000)
> Great depth of focus
> High resolution (<10nm)
> Can rotate specimen 
> View large, irregular specimens
22
Q

How must SEM samples be prepared?

A

Similar to TEM but w/ robust specimens

Delicate specimens need to be dehydrated - critical point drying

23
Q

Describe the critical point drying process

A
  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
24
Q

What are the problems with critical point drying?

What can be used to overcome these issues?

A

Shrinkage
Removal of substances soluble in organic solvents

Cryo-SEM
(Flash freeze in liquid N)

25
Q

Why is Cryo-SEM good?

A

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

26
Q

What are the 3 chemicals used during fixation?

A

Glutaraldehyde
Osmium tetroxide
KMNO4

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