Extra EM Techniques

Question 1

STM

Answer 1

Scanning Tunnelling Microscope
Res ~ 0.1nm

Quantum mechanical tunnelling effect > e- tunnel through a vacuum barrier between a sharp tip and the surface > creates a CURRENT that maps surface topography!

only suitable for conductive samples

Question 2

AFM

Answer 2

Atomic Force Microscope

Physical cantilever with sharp tip in end to measure the forces between atoms in tip, and in sample
BOTH conductive (electrode surfaces) and Non-conductive (proteins/DNA) materials!

High-res imaging (topographical mapping)
Force spectroscopy (interfacial forces)
Material property characterisation (nanoscale material properties adhesion, friction, stiffness, etc.)

Question 3

AFM modes

Answer 3

Contact mode
- tip constant contact w surface
1) constant height > scanner height fixed, deflection>topo
2) constant force > feedback loop adjusts scanner height to maintain constant deflection
- best for hard, can damage soft

Non-contact mode
- tip oscillates above the surface
- oscillation amplitude is kept constant via feedback loop
- protects samples, but lower res, requires stable conditions

Tapping (intermittent) mode
- hybrid of above
- oscillates, but taps surface at bottom of each swing
- oscillation amplitude is kept constant via feedback
- high res

Question 4

AFM applications

Answer 4

• nanomechanical properties, (resistance to deformation, stiffness)
• normal force + friction measurements in hydrogels (want low friction, long lasting gels)
• HIGH SPEED TYPE: video-rate, produces 50 frames per second, visualise dynamic behaviours at interfaces

Question 5

CryoEM overcomes what EM issues?

Answer 5

• possible to image hydrated/liquid samples
• stabilise beam-sensitive samples
• samples preserved closer to native/natural state
• structural, elemental, crystallographic data

Question 6

SPA

Answer 6

Single Particle Analysis
- reveals 3D structure of small proteins and macromolecules down to a few Angstrom resolution

• alternative to x-ray cryst. and NMR > these tehcniques require small molecules/for it to be crystallised and no good for large or enzymes

Question 7

CHALLENGES AND SOLNS OF CLEM

Answer 7

1) Sample compatibility {do OM first, EM second}
2) Sample prep {gridded sample holders, motorized microscope stages, find 3 points and triangulate}
3) photons are not electrons {ph travel in straight lines, e- spiral, un-twist the e- by calibrating distortion}
4) resolution gap {use deconvolution, undo “point spread” caused by optics}
5) navigating the cell {identify the cut depth to find the TEM section that matches the optical segment}