Atomic Force

Microscopy

PROTEOMICS
DIVYA GARG
M.Tech
6016001312

Atomic Force Microscopy

(AFM)
Family-

Scanning Probe Microscopy

(SPM) in 1981 by G. Binning and H.
Rohrer.

SPM-

sharp probe scanned across a

surface and some probe-sample
interaction(s) monitored

AFM

senses inter-atomic forces

that occur b/w a probe tip and a

History of AFM
First

by Gerd Binning and Cristoph Gerber

in 1985
Constructed by gluing tiny shard of diamond
onto one end of tiny strip of gold foil
End of the tip pressed against sample
surface
Sample scanned by tracking deflection of
cantilever by monitoring tunneling current to
2nd tip position above cantilever
Developed in order to examine insulating
surfaces

How it works AFM

experiment

Parts of AFM (Simple

schematic)
1.
2.
3.
4.
5.
6.
7.
8.

Laser
Mirror
Photodetector
Amplifier
Register
Sample
Probe
Cantilever

Basics of AFM
instrumentation
Tip

moves over the sample surface

Laser

light reflects from the surface

of cantilever

Deflection
Singles

in laser light is scanned

are generated and analyzed

Challenges of AFM regarding

design
Requirement

of sharp probe for high

resolution
Force b/w probe & sample should be
1 nN or less
Feedback controller should have a
rapid control
High speed computer that can
generate the images in real time
Vibration free stage

Imaging Modes

Contact mode
Measures

repulsion between tip and

sample
Force of tip against sample -->
remains constant
Feedback regulation keeps cantilever
deflection constant
Voltage required indicates height of
sample
Problem : Excessive tracking forces
to sample

Non- contact mode

Measures

attractive forces Van der

Waals forces b/w tip and sample
No touch
Cant use with samples in fluid
Used to analyze semiconductors
Doesnt degrade or interfere with
sample- better for soft samples

Tapping or Intermittent
contact mode
Tip

vertically oscillates b/w contacting

sample surface and lifting off at frequency 50,000 to 500,000 cycles/sec.

Oscillation

amplitude reduces as probe

contacts surface due to loss of energy

Advantages: Overcomes

problems of friction, adhesion

and electrostatic forces
More effective for larger scan size

Topography Imaging

Interpretation of forcedistance curve

Sample preparation
Suitable

substrate:
Flat and rigid (Mica, SiO2, stripped
Au)
Immobilization
Typical

of sample

sample size

Imaging of isolated molecules in air

Images of molecule
surface (2D)

Imaging of cells

Advantages of AFM
Sample

preparation minor, over a large range of

temperatures and in repetitive studies

High

resolution (measures forces as low as 10 pN)

allows topographical imaging of samples such as DNA
molecules, proteins absorption or crystal growth and
living cells absorbed on biomaterials etc.

Complementary

techniques for surface properties,

e.g. stiffness, hardness, friction or elasticity

Tool

for controlled mechanical nano-stimulation and

manipulation

Applications
Study

of unfolding of proteins

Imagining

of biomolecules

Antibody-

antigen binding studies

Ligand-

receptor binding studies

Binding

forces of Complimentary DNA strands

DNA-protein
Proteins

interaction

and liposomes

Applications
Imaging
Study
Ion

of cells

Surface frictional forces

channel localization

Quantification

of molecular interactions in
biological systems

Quantification of electrical surface charge

Limitations
Image

max ht. of the order 10-20 m

and a max scanning area of abt. 150x150 m
sq.

Scanning
Highly

speed is also limitation

dependent on AFM probes

Conclusion
AFM versatile tool to investigate:
- topography of surfaces
- properties of surfaces
- properties of single molecules
- forces within molecules

Protein folding

 Contd

Examples (further
reading)

References:
Nano

Science and technology consortium; NSTC

Best

R, Fowler S et al.; Cambridge University

Dept of Chemistry, MRC Centre for Protein
Engineering.

Kronenberger

science.

A.; School of Engineering and