microscopy is considered today the most powerful and versatile microscopic
technique to study various samples at nanoscale. It is a kind of scanning probe
microscopy. This technique makes use of the instrument named “Atomic Force
Microscope” for the real-time observation of given sample e.g. microbial cells surfaces
can be visualized more clearly than electron microscopic strategies under this
latest microscopic technique.
The versatility of
this technique lies in the fact that it cannot only generate the image in 3D
topography, but it can also provide many kinds of surface measurements to
scientists and engineers. The technique is very powerful in this way that the
images generated by AFM have greater atomic resolution regarding height
information along with least sample preparation.
microscope was initially designed to find out the topography of some
non-conductive samples. Later, the instrument is enhanced in many ways to
measure different other physical parameters e.g. surface hydrophobicity,
molecular interactions and surface charges (Yves F. Dufreˆne, 2002).
microscope was invented by collaboration of IBM (International Business
Machines) and Stanford University by a scientist named Binning in 1982 and in
1986, the first experimental implementation was made. He got a Nobel prize in
1986 (Surena Vahabi et.al. 2013).
process is performed not by the means of an incident beam as used by other
classical microscopies, but it works on the principle of sensing the forces
between the sample surface and a very sharp probe (Yves
F. Dufreˆne, 2002). The probe is basically known as “AFM sensor” or “AFM
tip” attached with a cantilever. The various detection methods are there based
on different types of motion of cantilever. The cantilever is deflected as the
tip is brought in the proximity of sample, due to the forces of attraction
between tip and sample. The deflection
of cantilever is measured by means of Hooke’s law.
(General principle of AFM)
(Instrumentation) of Atomic Force Microscopy:
The AFM consists of following main
AFM probe which is a sharp tip attached to a
Optical lever which measures the deflection
A feedback loop which allows to monitor the
interacting forces between the molecules on the cell surface with the ones that
are present on the tip.
A piezoelectric scanner which moves the tip in
a three-dimensional pattern relative to the sample surface.
A computer using as conversion system for raw
data obtained by the AFM instrument into an image or some other useful display
(UrošMaver et.al, 2016).
Mechanism of action of Atomic force
consists of a cantilever (a plate or beam attached to a support at one end)
made of silicon or silicon nitride having a sharp tip called probe. When the cantilever tip is brought near sample the
forces between them lead to the deflection of cantilever. This force can be
measured by Hook’s Law.
Where F is force, k is spring constant and x is
Most of AFM nowadays uses optical system laser beam
detection system called photodiode. The hardness is recorded
visually and can be pictured on the computer in real time.
experiences the Repulsive Van der Waals
forces when it is near the sample (Contact
mode) and Attractive Van der Waals
forces when probe is far away from the sample. (Non-contact mode).
Primary Imaging Modes in AFM:
the contact mode the separation between sample and probe is less than 0.5nm.
The force acting on the tip is Repulsive and the feedback loops maintained
constant force between sample and tip.
In the non-contact mode, the separation
between sample and probe is 0.1-10 nm. The force acting on the tip is
Attractive and the feedback loop monitors the changing in amplitude.
the tapping mode the separation between sample and probe is 0.5-2 nm. The
cantilever oscillates at resonant frequency and slightly taps on the surface
making contact and feedback loop maintains the constant oscillation amplitude.
easily be prepared, and 3D image is obtained.
Work in dynamic
environments i.e. air, vacuum or liquid
information about Height
systems can easily be studied
only image a maximum height on the order of 10-20 micrometers and a
maximum scanning area of about 150×150 micrometers.
scanning speed of AFM is less.
of the piezoelectric material can affect the imaging of AFM.
Tip or sample can occur.
In cell Biology:
distinguish between normal and abnormal cells and Interactions among the
In Molecular Biology:
interaction and Ligand-Receptor interaction study
forces present between complementary DNA strands and DNA condensation
increases the stiffness of cardiac myocytes, which can be measured with the
Nano indentation of the AFM.