Principles of Atomic Force Microscopy
There are several techniques that allow to study the properties of living cells (i.e., their ability to deform and to adhere) at the single cell and molecular levels. Among them, the noteworthy ones are those delivering information of local character, such as atomic force microscopy (AFM, ). In recent years, an ample evidence has demonstrated the functionality of this technique to characterize changes of single, living cells—based on either their elastic properties or single molecule interactions in respect of alterations in cellular functionality, structure, etc. This chapter introduces basic operational aspects of atomic force microscopy.
Principles of the AFM Operation
The idea of the AFM operation is very straightforward, but its realization requires highly advanced technology. A sharp, delicate probing tip, mounted at the end of a compliant cantilever, is moved in close proximity over an investigated surface. The probing tip senses forces acting within the contact area. These forces cause the deflection of a compliant cantilever that is monitored and recorded by a detector system. Independently of the technical details of AFM construction, there are three basic
Cellular Analysis by Atomic Force Microscopy Malgorzata Lekka
Copyright © 2017 Pan Stanford Publishing Pte. Ltd.
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elements common for all types of apparatus: (i) a cantilever with a probing tip, (ii) a system detecting the cantilever deflection, and (in) a system that provides scanning and positioning.
Figure 3.1 presents a scheme of the AFM working in liquid conditions, where the use of a so-called “liquid ceil” is strongly needed. The scheme shows also the most common cantilever detection system, composed of a laser and a position-sensitive photodetector (i.e., photodiode). Initially, a laser beam is focused at the free end of a cantilever. Then, in a close proximity to the investigated surface, interaction forces cause the cantilever deflection, thus moving the spot position of the reflected laser beam within an active area of the photodiode. The position of the laser beam spot, delivering the information on the cantilever displacement z, can be converted into force F using Hooke’s law:
where kcant denotes the cantilever spring constant, expressed in N/m.
Figure 3.1 Basic elements of the atomic force microscope using optical detection system. Independently of the AFM device type, there are three basic elements, namely: a cantilever with a probing tip, and two systems—one that detects cantilever deflection, and the other one that provides scanning and positioning (since the principle of operation is not dependent on the environment, i.e., ambient conditions or liquid, a “liquid cell” setup is schematically shown).
The sample, attached strongly to a support (like mica or glass coverslip), is mounted on a holder fixed to a piezoelectric scanner. The scanner provides the possibility to perform a raster scan over the investigated sample surface. Usually, the piezoelectric scanner is mounted on top of the coarse positioning system facilitating a convenient and rapid sample exchange.