Atomic Force Microscopy-Based Force Spectroscopy …
587
predominant capillary forces. Inside fluid cell biological samples like microbial or
mammalian cells can be submerged in a desired buffer or media to maintain the
physiological condition of the cell minimizing the capillary forces.
Although force spectroscopy is very sensitive technique, but it needs to be
performed with outmost precaution to get reproducible results.
2 Definition and Concept
Accurately and precisely measuring the forces associated with several biological
processes is very important for their understanding. To investigate these intricating
biological processes based on probing forces, single molecule force spectroscopy
plays a crucial role. Single-molecule force spectroscopy can be recorded using several
other manipulation techniques like optical and magnetic tweezers, AFM technique,
microneedle manipulation [4], biomembrane force probe [5], and flow-induced
stretching [6]. Optical tweezers, magnetic tweezers and atomic force microscopy
(AFM) are the most commonly used single-molecule interaction probing technique
that can operate on the length scale of 10
–10 to 10
–4 m and the force range of 10
–15
to 10
−8 N. In this chapter we will focus on AFM based force microscopy (AFM-FS)
and single molecule force spectroscopy (AFM-SMFS).
This spectroscopy precisely measures the inter- and intramolecular forces operating between the tip and sample, with piconewton resolution. This unique ability
of AFM enables the identification of certain interactive forces at single molecule
level, owing to its very precise sensitivity towards the identification of the force.
Further, AFM-SMFS generally uses functionalized tips with single molecule or a
tiny group of probe molecules which precisely detect a particular target molecule on
the surface of sample. In force spectroscopy, raster scanning of the sample surface is
not required like imaging rather the tip may come into the contact with the surface
and then retracted. The spectroscopy mode allows the cantilever movement along
the vertical direction only (z axis) so that it can move toward the surface and then
retract minimizing any xy movement. During this procedure, the cantilever deflection
with respect to vertical displacement of the piezo scanner is recorded. Generally, the
raw data obtained is represented in mV. In order to convert it to nm unit deflection
sensitivity of the cantilever need to be calculated on hard surface before actual experiment. The deflection sensitivity of the cantilever can be calculated by extracting a
parallel line to the 2–4 segment of a typical force plot curve as shown in Fig. 2.
The deflection sensitivity measurement needed the calibration for hard surfaces so
that no indentation occurs (zero indentation depth). Each of the cantilever exhibits
different deflection sensitivity which depends on the cantilever length, thickness,
coating, and contamination. Besides this, the laser light position on the cantilever
and the laser beam reflection (spot size, shape) quality from the cantilever governs the
sensitivity of cantilever. This deflection sensitivity, which is a very crucial parameter
can be defined as the minimum deflection required to produce an observable change
in photodetector (x/mV ). Using deflection sensitivity raw data or curve can be
587
predominant capillary forces. Inside fluid cell biological samples like microbial or
mammalian cells can be submerged in a desired buffer or media to maintain the
physiological condition of the cell minimizing the capillary forces.
Although force spectroscopy is very sensitive technique, but it needs to be
performed with outmost precaution to get reproducible results.
2 Definition and Concept
Accurately and precisely measuring the forces associated with several biological
processes is very important for their understanding. To investigate these intricating
biological processes based on probing forces, single molecule force spectroscopy
plays a crucial role. Single-molecule force spectroscopy can be recorded using several
other manipulation techniques like optical and magnetic tweezers, AFM technique,
microneedle manipulation [4], biomembrane force probe [5], and flow-induced
stretching [6]. Optical tweezers, magnetic tweezers and atomic force microscopy
(AFM) are the most commonly used single-molecule interaction probing technique
that can operate on the length scale of 10
–10 to 10
–4 m and the force range of 10
–15
to 10
−8 N. In this chapter we will focus on AFM based force microscopy (AFM-FS)
and single molecule force spectroscopy (AFM-SMFS).
This spectroscopy precisely measures the inter- and intramolecular forces operating between the tip and sample, with piconewton resolution. This unique ability
of AFM enables the identification of certain interactive forces at single molecule
level, owing to its very precise sensitivity towards the identification of the force.
Further, AFM-SMFS generally uses functionalized tips with single molecule or a
tiny group of probe molecules which precisely detect a particular target molecule on
the surface of sample. In force spectroscopy, raster scanning of the sample surface is
not required like imaging rather the tip may come into the contact with the surface
and then retracted. The spectroscopy mode allows the cantilever movement along
the vertical direction only (z axis) so that it can move toward the surface and then
retract minimizing any xy movement. During this procedure, the cantilever deflection
with respect to vertical displacement of the piezo scanner is recorded. Generally, the
raw data obtained is represented in mV. In order to convert it to nm unit deflection
sensitivity of the cantilever need to be calculated on hard surface before actual experiment. The deflection sensitivity of the cantilever can be calculated by extracting a
parallel line to the 2–4 segment of a typical force plot curve as shown in Fig. 2.
The deflection sensitivity measurement needed the calibration for hard surfaces so
that no indentation occurs (zero indentation depth). Each of the cantilever exhibits
different deflection sensitivity which depends on the cantilever length, thickness,
coating, and contamination. Besides this, the laser light position on the cantilever
and the laser beam reflection (spot size, shape) quality from the cantilever governs the
sensitivity of cantilever. This deflection sensitivity, which is a very crucial parameter
can be defined as the minimum deflection required to produce an observable change
in photodetector (x/mV ). Using deflection sensitivity raw data or curve can be