1. Basic Electron Microscopy
Arthur Rowe
The Knowledge Base at a Simple Level

2. Introduction
These 3 presentations cover the fundamental theory of electron microscopy
In presentation #3 we cover:
requirements for imaging macromolecules
aids such as gold-labelled antibodies
the negative staining method
the metal-shadowing method
Including high-resolution modifications
vitritied ice technology
examples of each type of method

3. requirements for imaging macromolecules
sufficient CONTRAST must be attainable, but
> bio-molecules are made up of low A.N. atoms
> & are of small dimensions (4+ nm)
> hence contrast must usually be added
sufficient STABILITY in the beam is needed
> to enable an image to be recorded
> low dose ‘random’ imaging mandatory for any
high resolution work

4. ways of imaging macromolecules
ADDING CONTRAST (with heavy metals)
> negative contrast
+ computer analysis
+ immunogold labels
> metal shadowing
+ computer enhancement
USING INTRINSIC CONTRAST
> particles in thin film of vitrified ice
+ computer acquisition & processing

5. ways of imaging macromolecules
using immunogold labels to localise epitopes
> widely used in cell biology
> beginning to be of importance for macromolecules
Au sphere
Mab
epitope
macromolecule

6. negative staining
particles
Electron dense negative stain

7. negative staining
• requires minimal interaction between particle & ‘stain’
to avoid binding, heavy metal ion should be of same charge +/- as the particle
positive staining usually destructive of bio-particles
biological material usually -ve charge at neutral pH
widely used negative contrast media include:
anionic cationic
phosphotungstate uranyl actetate/formate
molybdate (ammonium) pH ~ 4)

8. metal shadowing - 1-directional

9. metal shadowing - 1-directional
Contrast usually inverted to give dark shadows
> resolution nm - single 2-fold a-helix detectable
- historic use for surface detail
- now replaced by SEM
> detail on ‘shadow’ side of the particle can be lost
> apparent ‘shape’ can be distorted
> problems with orientation of elongated specimens
- detail can be lost when direction of
shadowing same as that of feature
> very limited modern use for macromolecular work

10. metal shadowing - rotary

11. metal shadowing - rotary
Contrast usually inverted to give dark shadows
> resolution nm - single DNA strand detectable
- historic use for ‘molecular biology’
(e.g. heteroduplex mapping)
> good preservation of shape, but enlargement of
apparent dimensions
> in very recent modification (MCD - microcrystallite
decoration), resolution ~1.1 nm

12. particle in vitrified ice: low contrast
particles examined at v. low temperature, frozen in a thin layer of vitrified (structureless) ice - i.e. no contrast added

13. particle in vitrified ice: low contrast
average of large numbers (thousands +) of very low contrast particles enables a structure to be determined

14. particle in vitrified ice: low contrast
average of large numbers (thousands +) of very low contrast particles enables a structure to be determined:
resolution may be typically 1 nm or better
this is enough to define the “outline” (or ‘envelope’) of a large structure
detailed high resolution data give us models for domains (or sub-domains) which can be ‘fitted into’ the envelope
ultimate resolution of the method ~0.2 nm, rivalling XRC/NMR

15. particle in vitrified ice: the ribosome

16. particle in vitrified ice: phage T4 & rotavirus

17. case study : GroEL-GroES
• important chaperonins
hollow structure
• appear to require ATP (hydrolysis ?) for activity

18. particle in vitrified ice: low contrast
the chaperonin protein GroEL visualised in vitrified ice
(Helen Saibil & co-workers)

19. GroEL GroEL + ATP GroEL+GroES +ATP

20. DLS as a probe for conformational change in GroEL/ES

21. GroEL GroEL + ATP GroEL+GroES +ATP

23. case study : pneumolysin
• 53 kD protein, toxin secreted from Pneumococcus pneumoniae
• among other effects, damages membrane by forming pores
• major causative agent of clinical symptoms in pneumonia

24. electron micrographs of pores in membranes caused by pneumolysin
RBC / negative staining membrane fragment metal shadowed

25. Homology model based upon the known crystallographic structure of
Pneumolysin
Homology model based upon the known crystallographic structure of
Perfringolysin

26. Pneumolysin - homology model ± domain 3, fitted to cryo reconstruction

27. Pneumolysin - EM by microcrystallite decoration (MCD) reveals orientation of domains

28. identified within the oligomeric form (i.e. the pore form)
Pneumolysin
- monomers
identified within the oligomeric form (i.e. the pore form)

29. case study : myosin S1
• motor domain of the skeletal muscle protein myosin
• 2 S1’s / myosin, mass c. 120 kD
• ‘cross-bridge’ between myosin and actin filaments, thought to be source of force generation

30. myosin is a 2-stranded coiled-coil protein, with 2 globular (S1) ‘heads’
S1 unit

31. Each S1 unit has a compact region, & a ‘lever arm’ connected via a ‘hinge’ to the main extended ‘tail’

32. Myosin S1 imaged by Microcrystallite Decoration (no nucleotide present)

33. Effect of nucleotide (ADP) on the conformation of myosin S1 as seen by MCD electron microscopy

34. case study : epitope localisation in an engineered vaccine
• a new vaccine for Hepatitis B contains 3
antigens, S, S1 & S2, with epitopes on each
• but does every particle of ‘hepagene’
contain all 3 of these epitopes ?
• Mabs against S, S1 & S2 have been
made & conjugated with gold:
S 15 nm
S1 10 nm
S2 5 nm

35. immunolabelling of one epitope (S1) in hepagene using 10 nm-Au labelled Mab

36. triple labelling of 3 epitopes on hepagene

37. Basic Electron Microscopy
Arthur Rowe
End