DNA Imaging Techniques

Question 1

Principles of AFM

Answer 1

• atomic force microscopy
• cantilever obeys Hooke’s law, F=kz
• like a sharp tip on a spring
• behaves very well as a simple harmonic oscillator when excited: fo=1/2π √[k/m]
• each mode of vibration at temperature T holds kbT/2 of energy
• higher modes are harder to excite leading to a lower amplitude
• can use static deflection or amplitude of the catilever to detect surface for imaging

Question 2

Where does AFM work?

Answer 2

• in all environments (vacuum, air, liquid)

- resonant properties of the cantilever are effected by the surrounding medium quality factor, Q

Question 3

What are the two ways to image with AFM?

Answer 3

• cantilever deflection, direct contact medium

- amplitude reduction, tapping mode

Question 4

AFM

Direct Contact Mode

Answer 4

• continuous tracking force from cantilever
• causes damaging shearing forces
• capillary force in air so
• not usually used for biological samples

Question 5

AFM

Tapping Mode

Answer 5

• excite cantilever at one frequency as it reaches the surface of the object the amplitude changes
• force is always normal to the surface which eliminates any damaging shearing forces

Question 6

AFM of DNA Systems

Answer 6

• good signal to noise ratio for single molecule resolution
• no contrast agents required
• hydrating environments (can use in liquid or air, the water vapour in air is enough to keep DNA in B form)
• 3D information, topography and topology

Question 7

Muscovite Mica

Answer 7

• crystalline structure, layered structure
• when cleaved it leaves an atom smooth surface microns in size
• put water on this surface and it will spread out due to exposed oxygens on the crystalline surface
• if you then add proteins or other molecules, they will spread out on the water film
• stick them down, then perform AFM

Question 8

AFM vs STM

Answer 8

• STM is sensitive to most loosely bonded electrons with energy near the fermi level
• AFM responds to all electrons including the core electrons
• since fermi electrons are spatially less confined, AFM should be able to achieve a higher resolution than STM

Question 9

Single Atom AFM

Answer 9

-since AFM can detect core electrons, in principle it could have atomic or even sub-atomic resolution

Question 10

Can you perform AFM and STM at the same time?

Answer 10

• if you have an AFM cantilever with a charged tip, you can simultaneously perform AFM and STM
• the molecular/atomic species ending the tip also has an influence on resolution

Question 11

STM Tunnelling Mechanism

Electrons Tunnel to Sample

Answer 11

• electrons tunnel from the lowest occupied energy states to the highest unoccupied energy states around the Fermi energy
• altering the bias voltage can access different electron energy states in the sample or the tip

Question 12

STM

Current vs Distance

Answer 12

• tunnelling exponentially depends on distance
• it is easier to get close to the surface as you know that an increasing current means that you are getting closer
• single atom tip, a large proportion of current is tunnelling through the front atom

Question 13

AFM

Current vs Distance

Answer 13

• tip-sample interaction is close to a Lennard-Jones potential
• interaction volume between the tip and the sample is larger than STM
• it is harder to get close to the surface than with STM as for AFM the potential is not monotonic
• STM is high resolution on smooth surfaces but quickly deteriorates on rougher surfaces

Question 14

Topology

Definition

Answer 14

-how things are connected

Question 15

Topography

Definition

Answer 15

-surface roughness