Atomic Force Microscope and its potential use in biology

- Sudhir K Shukla

Journal club presentation

Atomic Force Atomic Force Microscope and its Microscope and its

potential use in biologypotential use in biology

Scanning Probe MicroscopyScanning Probe Microscopy

STM: scanning tunneling microscope

tunneling of electrons between probe and surface

AFM: atomic force microscope

measuring of the force on the probe tip

OFM: Optical force microscopemeasuring of the force on the optically trapped particle

MFM: magnetic force microscopeAFM with magnetical probe

yx


nA

R

piezo-element

e-e-

e-

e-e-

e-

e-

e-

e-

< 1nm

tunneling of electrons through air between probe

and surface

only conducting material

probe

x-y stage


Icontrol

piezo-element (changes length at different voltages)

nAItip

∆I

R

∆I -> ∆Vtransfer

Challenges of the STM

1. Works primarily with conducting materials 2. Vibrational interference

3. Contamination

• Physical (dust and other pollutants in the air)

• Chemical (chemical reactivity)

AFM: Atomic Force MicroscopeAFM: Atomic Force Microscope

• The AFM brings a probe in close proximity to the surface

• The force is detected by the deflection of a spring, usually a cantilever (diving board)

• Forces between the probe tip and the sample are sensed to control the distance between the the tip and the sample.

van der Waals force curve

AFM probe scans over the surface e.g. living cells,

chromatin fibers

laser photodiode

piezo-element

probe

AFM: how it works

feedback

AFM: how it works

cantilever tip

laser

cantileverpiezo

y

z

x

photodiode

Scanning the SampleTip brought within nanometers of the sample (van der Waals)

Radius of tip limits the accuracy of analysis/ resolution

Stiffer cantilevers protect against sample damage because they deflect less in response to a small force

Scanning Modes1. Contact (Repulsive force)At short probe-sample distances, the forces are repulsive

2. non-contact (Attractive Force )

• At large probe-sample distances, the forces are attractive

• The AFM cantilever can be used to measure both attractive force mode and repulsive forces.

3. Tapping mode (vibrating mode)• Better resolution • Minimal damage to sample


1. Contact Mode Contact mode operates in the repulsive regime of the van der Waals curve

Tip attached to cantilever with low spring constant (lower than effective spring constant binding the atoms of the sample together).

In ambient conditions there is also a capillary force exerted by the thin water layer present (2-50 nm thick).


2. Non-Contact Mode

Uses attractive forces to interact surface with tip

Operates within the van der Waal radii of the atoms

Oscillates cantilever near its resonant frequency (~ 200 kHz) to improve sensitivity

Advantages over contact: no lateral forces, non-destructive/no contamination to sample, etc.


3. Tapping mode

Change in amplitude measured

Change in phase measured

Biological Applications

1. Study Unfolding Of Proteins

2. Imagining Of Biomolecules

3. Force Measurements In Real Solvent Environments

4. Antibody-Antigen Binding Studies

5. Ligand-Receptor Binding Studies

6. Binding Forces Of Complimentary DNA Strands

7. Study Surface Frictional Forces

8. Ion Channel Localization

path of AFM tip

AFM tip

superhelical DNA plasmid

DNA double helix

Mg2

+

negatively charged mica surface

Mg2

+

Mg2+Mg2+Mg2+Mg2+

movement of the AFM tip along the sample

AFM image of a 6.8 kb superhelical plasmid

AFM tip

Molecular Force Probe

•Functionalizing Cantilevers as a live biological substrate

• Receptor binding in native environment can be observed

Tkk

xr

x

Tkf

Boff

fBm 0

ln

Panorchan, P. et al. Journal of Cell Science, 119. 2006

Functionalization of AFM Tip

Imaging live cells: from structure to function

Single molecule imaging

Molecular recognition maps demonstrating that clustering of theyeast sensor Wsc1 (green) is strongly enhanced by hypoosmotic shock

Buffered solution Deionized water

Single molecule imagingMolecular recognition maps documenting the distribution of single Als5p adhesins (red) on a single yeast cell.

Unfolding studies of spectrin

C-terminal

Helix C

N-terminal

Helix B

Helix A

molecule that contributes to the mechanical properties,

especially the elasticity of the cells

measurement of its mechanical stability provides information

about the physiological function

Stretching spectrin with an AFM

distance

forc

e

1 2 3

surface

cantilever tip

4 repeated spectrin domain

1. adhesion force between cantilever tip and surface

2. dissociation from the folded state to the intermediate unfolded state

3. dissociation from the intermediate to the total unfolding state

0.15

100 30 40

0.05

20 50

0.10

0.20

0.00

unfolding force (pN)

pro

babili

ty

stretching spectrin with an AFMfo

rce (

pN

)

0 20 40 60 80

0

4020

608010

0

distance (nm)

-20

MFM: magnetic force microscope

AFM with magnetic probe

e.g. hard disc, tape

magnetic tip

laser photodiode

piezo-element

Thanks