Post on 24-Dec-2014
description
- 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
STM: scanning tunneling microscope
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
STM: scanning tunneling microscope
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
van der Waals force curve
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).
van der Waals force curve
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.
van der Waals force curve
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