Scanning force microscopy (SFM) which

is also know atomic force microscopy is

one of the scanning probe microscopic
techniques which provides a very high-
resolution and great details of the
samples.
This technique provides the images by
scanning the sample in a raster pattern
and produces extra ordinary results. The
data is gathered by sensing the surface
with a mechanical probe.
The construction of an AFM consist of a
cantilever which supports the tip, a
system for detecting cantilever deflection with a feedback loop, and a scanner to control the
relative position of tip and specimen.
Cantilever is typically made of silicon or silicon nitride with a tip radius of curvature on the order of
nanometers. When we bring the tip closer to the sample the forces present between the tip of the
probe and the surface causes the deflection of cantilever .forces that are measured in AFM
include mechanical contact force, van der Waals forces, capillary forces, chemical bonding,
electrostatic forces, magnetic forces and some other forces which certainly depends upon the
situation.














Figure 2. The principal components of an AFM

Figure 1. AFM Construction
Cantilever deflection plays a vital role during the AFM operation, for a given deflection of the
cantilever the force applied normal to the surface will depend on the bending stiffness or spring
constant, k, given in units of N/m. The spring constant for a single-beam cantilever depends on the
geometry of the lever and the material modulus.

k =
Lwt
3
4I
3
Equation 1
Where:
w = width
l = length
E = modulus
t = thickness
Scanner is a back bone of any scanning probe microscopic technique. They are responsible for
controlling the position of probe according to the shape of specimen with extreme precision.
Scanners are made of ceramic material like zirconate titanate and they possess a very unique
property of piezoelectricity effect an, electric field is applied and the ceramic changes shape.









Figure 3. Piezo scanners the tripod (left) and tube (right)


In scanning probe microscopy the basic principle revolves around the detection and measuring
the forces between the tip and surface.
Therefore it is necessary to have good understanding of the interaction forces between two solids
at small distances. The usual first step is to consider the interaction between two isolated atoms .
A good approximation to the energy of this interaction is the Lennard-Jones potential. This is
expressed below.
u(r) =
A

12
-
B

6
Equation 2

And the force between the atoms is given by :
F(r) = -
d0
d
=
A

13
-
B

Equation 3
Positive forces repel, and the first term governs at short distances. The negative term is the van
der Waals force that attracts and dominates at relatively large distances. For an AFM with an
ideally sharp tip it might be better to consider the interaction of an atom with a plane.

Figure 4. A Lennard-Jones potential between two atoms and the associated force between them

AFM can be operated in two general modes, details of which are described below. In our
experiment we used tapping mode for the investigation of DNA structure.

The contact is the simplest one. In this mode the set point for any net force is chooses for which
the tip of probe will be in contact with the surface of the specimen.

This mode is used quite frequently and one of the useful mode while investigating polymers. In
Tapping Mode-AFM cantilever with stiffness typically 5-50N/m is driven into oscillation using a
bimorph piezo driver at its base.

Noncontact AFM technique was introduced in 1991 . This mode is preferred for electrically
conductive polymers for high-resolution scanning images at low amplitude.


We conducted our study on DNA specimen using tapping mode Atomic force microscopy.
Two samples were produced , reason behind was to avoid time wastage in case of sample
damage. Mice plates were exfoliated using adhesive tape and then a drop of DNA was deposited
on mica plate . After a while we washed the mica plate with care and samples were ready for
characterization phase.




Scanning probe microscopy techniques have shown great results in biological studies and AFM
has proven to be a useful tool to observe DNA conformations, substructures and protein-DNA
complexes. A huge advantage of AFM which makes it unique from the other analytical technique
is its versatility. One of the most appealing features is that it can be operate in aqueous
environments, due to which we can mimic the physiological conditions to study the DNA structure.
Model used to investigate the DNA molecule contour length was Worm-Like model. In this model,
the DNA is treated as a flexible rod of length L that curves smoothly as a result of thermal
fluctuations.
The DNA chain is distributed into small segments and then small segments are added together to
calculate the contour length. This critical job in done by the software, the procedure of software
evaluation is described below.
The mathematical expression for calculating contour length is given in equation 1.
R
N
I
1
2
Equation 4

Where
R = Average end to end distance






Figure 5. Image of DNA taken from AFM
By using Nanoscope program first we imported the images to this program. Images were scaled
the images and by using the polyline command from the tool bar, we draw a polyline along the
DNA molecules. But while choosing DNA molecule for analysis we certainly have to make sure
that it is not coiled up, or it is also not entangled with other DNA molecules in the surrounding.
Otherwise overlapping of DNA will lead to inaccurate results since we are interested and meant to
measure the contour length which is a fully extended length of a single DNA molecule.

As we finished selecting and drawing the polylines long the DNA molecules, then we switched to
the analysis software called CHAINSTAT. This software runs on LINUX. By using the commands
from the manual, subsequently we got the analysis result files in the form of 00 .dat file. Result
files were exported to Origin program and further investigations were carried out to measure the
contour length and the histogram against the occurrence frequency.

Table 1. Contour lengths
Sr. No Contour Length (nm)
1 447.4508
2 530.7697
3 579.0375
4 747.4139
5 665.8646
6 737.2597
7 364.094
8 776.8804
9 670.7185
10 576.1659
11 906.1692
12 688.4338
13 782.5585
14 548.0685
15 592.4421
16 463.1318
17 475.8434
18 556.131
19 463.1886
20 731.8441
21 279.6571
22 478.296
23 589.0529
24 471.1278
25 515.7656
26 594.8688
27 587.3974
Average 585.9123
The Average value of Contour length measured is 585.9123nm. If we analyze the contour Length
distribution in below Histogram we can conclude that the maximum occurrence of chain length is
in the range of 400nm to 500nm. There is no doubt that these measurements seems to be
deviating little bit, since a very accurate measurement should follow a Gaussian curve. But we do
certainly believe that the range described above does seems to be very close to the correct
measurement.















Figure 6. Histogram for contour length distribution
There are certain factors which can influence our result during AFM measurement. Following are
the most important and impactful.
Table 2. Possible errors
Sr .No Artifacts/ Error Source Type of Artifacts
1 Sample Preparation
Sample Related 2 Sample Environment (E.G., Humidity)
3 No. of Sample Being Measured
4 Scanner Motion
Instrumental
5 Tip Geometry
6 Noise (Mechanical, Acoustic, Or Electronic)
7 Drift (Thermal Or Mechanical)
8 Signal Detection
9 Improper Use Of Image Processing Analysis

During our experiment, our first attempt to find out uncoiled DNA chain was not successful and the
possible reason was associated to sample preparation. One very key element during sample
preparation is the use of buffer solution which uncoils the DNA chains. Since the properties of
biological substances vary significantly with small changes in pH, they require environments in
which the pH is insensitive to additions of acids or bases.

The contour length distribution will vary a lot if the no of samples are lower in number. To obtain a
good statistical results, high no of samples are required to me analyzed and measured their
contour length in that case we can find very good results and the chain length distribution curve
will also have Gaussian behavior since we analyzed only 27 sample which are pretty less.

Size of the probe and its geometry also influences the measurement results. The shape of the
probe and its tip is vital for detailed interpretation of AFM or scanning tunneling microscope (STM)
images. Tip size which is finite will cause an apparent increase in size of small protruding objects
and an associated apparent reduction in size of pits. Such artifacts are very common in SMP or
STM. If the surface features are all small or have a small slope, then only the very tip of the probe
is important, whereas an abrupt step might interact with the probe well away from the tip.



The determination of persistent length is calculated through the variance of the Gaussian
distribution of . Gaussian distribution of is described by the following equation:
o
2
=
I
P
=< 0
2
> Equation 5

For Worm-Like chain model the the angle distribution fulfills the following Gauss distribution:
(0) =
1
c2n
c
-0
2
2o
2
Equation 6

If we integrate Eq2 We will get error function:
(0) = ] (t)Jt =
1
c2n
] c
-
t
2
2o
2
Jt
0
-
0
-
Equation 7

From the equation 3 we can get the value of error function, however we can modify this equation
for inverse error function. This will be written as following
cr
-1
(2 (0) -1) =
0
2c
Equation 8

Now we can plot this inverse error function and can get the values of slope my using line equation
which says, y=mx+c (general expression).
From the slope of line we can reach to our goal of calculating the persistence length.
P = 278

Equation 9

Since the lengths of the drawn segments are in all direction so we need to normalize the tangent
angel .
Finally we obtain the expression of persistent length
6 28

Equation 10

Figure 7. Plot of the error function for overall number of angles.
6 28

Equation 11

b=slop= 16.498
Substituting the values in Eq 6,
6 2 9 16.498

6 544@A
P is our persistent length of the DNA molecule chain which is enormously greater than the
literature value of DNA molecule chain persistent length.
There can few errors which have led to this huge increase in the persistent length of DNA chain.
A greater possibility is that the DNA molecules chains are loops together or coiled and they are not
in equilibrium conformation.
Angle (rad)
E
r
r
o
r

F
u
n
c
t
i
o
n

Relaxed state of chain is very important when we are measuring the persistent length, because if
the chain is under stress it will behave stiffer and the angel between the two tangents which is
Infact the measure of persistent length will become difficult to be located . So if we have to go far
to find the angle means we will have a higher persistent length. Higher persistent length can be
the result of stiffness.



1) Polymer Microscopy Third Edition by Linda C. Sawyer, David T. Grubb, Gregory F. Meyers
2) Introduction to Polymer Physics by M. DOI
3) Lab Manual