Electron microscopy

Question 1

Resolution vs light microscopy

Answer 1

Much higher for elecron

However short wavelength of electrons means limit of resolution t

Question 2

Light wavelength and resolution

Answer 2

400-710 nm

200nm

Question 3

Electron wavelength and resolution

Answer 3

2. 5 pm

0. 1 nm

Question 4

2 types of EM

Answer 4

SEM and TEM

Question 5

What do 2 types have in common

Answer 5

both in a vaccuum
Both have electrons emitted by a filament
condenser lens focuses electron beam
but different positions of specimen

Question 6

TEM

Answer 6

electrons either scatter or hit fluorescent screen at the bottom

Question 7

SEM

Answer 7

Electrons focused on metal coated specimen, electrons from the metal are collected by detector

Question 8

TEM techniques: Direct examination

Answer 8

grids and formvar, contrast, replication

Question 9

Grids and formvar

Answer 9

samples placed on copper grid coated with formvar

Question 10

Contrast

Answer 10

Materials have light elements eg C, H, low electron scattering power so less contrast
Enhance contrast by staining with heavy metals eg lead, uranium
Non-specific
Contrast enhanced by shadowing

Question 11

Shadowing

Answer 11

heavy metal evaporated from a wire in a vaccum chamber casting shadow on adjacent sample

Question 12

Replication

Answer 12

Replicas made for fragile samples

Specimen coated with repl material eg thin film of evaporated carbon

Question 13

TEM techniques: sections from tissues

Answer 13

fixation, dehydration, embedding, sectioning, staining

Question 14

Fixation

Answer 14

Preserve sample as it was during sample prep
Using:
Glutaraldehyde (protein amino groups)
Osmium tetroxide (proteins and lipids)
Potassium permanaganate (membranes)

Fixatives crosslink molecules

Question 15

Dehydration

Answer 15

HIgh pressure and water content- sample will die so needs to be dehydrated
Incubate in varying concs of ethanol in 10% steps with gentle agitation

Question 16

Embedding

Answer 16

Place sample in BEEM capsule
Infiltration with unpolymerised epoxy-resin, Epon or Araldite
Polymerisation of resin in the capsule
Remove resin block

Question 17

Sectioning

Answer 17

ultra microtome (estimate section thickness by interference colours)
Sections picked up on formvar coated grids

Question 18

Staining

Answer 18

Fixatives vary in rate of penetration of tissues, ability to fix different tissues and can cause shrinkage/swelling of tissues

Question 19

Location of cell components by light microscopy

Answer 19

Histochemical dyes
Antibodies linked to fluorescein isothiocyanate (FITC)
GFP
Chromogenic enzyme substrate

Question 20

Location of cell components EM

Answer 20

Antibody linked to collodial gold: 2 sizes of gold- 5nm and 10nm- differentiate compounds
Enzyme localization by linking product to heavy metal eg lipases and Pb
Enzyme localization if products are electron dense

Question 21

SEM general points

Answer 21

electron beam scans specimen, causes it to emit electrons to give image
Specimen ca be rotated/tilited

Question 22

SEM preparation

Answer 22

Similar to TEM, but robust specimens

Question 23

Critical point drying- SEM

Answer 23

1. dehydrate in ethanol-water series
2. Replace with amyl acetate
3. Subject to liquid CO2 under pressure
4. Raise temp, liquid CO2 converts to gas leaving dry specimen

Question 24

Problems SEM

Answer 24

shrinkage, removal of substances soluble in organic solvents

Cryo-SEM overcomes these limitations

Question 25

Cryo-SEM

Answer 25

direct examination of frozen hydrated specimens
Flash freeze in liquid nitrogen
sample retains H2O
No exposure to fixatives or solvents, no heavy metal staining
Prep time short
3D image generated from computer
Allows examination of hydrated, unfixed and unstained specimens by maintaining them a few degrees above absolute zero
(LOW temp SEM)

Question 26

For TEM

Answer 26

Thick sections of a specimen must be fixed, dehyd, embedded in plastic and stained with heavy metals